Application of substances containing AuCs in preparing medicine for preventing and treating Alzheimer&#39;s disease

ABSTRACT

Disclosed are a pharmaceutical use of a gold cluster and a substance containing the gold cluster and the preparation method and use thereof. The gold cluster and substance containing the gold cluster can inhibit the aggregation of Aβ and α-syn, has excellent effects on the levels of cell models and animal models, and can be used to prepare drugs for preventing and treating Alzheimer&#39;s disease and/or Parkinson&#39;s disease.

FIELD OF THE INVENTION

The present invention relates to the technical field of nanometernano-drugs, particularly to substances containing gold clusters (AuCs)and preparation method and application thereof.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases are one of the major threats to human health.Their common pathological features are abnormal entanglement of proteinsand their amyloidosis in nerve cells, and associated neuronal apoptosisand neurological impairment. Alzheimer's disease (AD) and Parkinson'sdisease (PD) are the most typical two of them. The clinicalmanifestations of AD are characterized by memory and cognitivedysfunction and changes in personality and behaviors, while the clinicalmanifestations of PD mainly include static tremor, bradykinesia,muscular rigidity, postural gait disorder and other dyskinesia. Both ADand PD mainly occur among old people, and the incidence increases withage. Taking AD for example, the incidence among the people above 65 is5%, but above 30% among the people above 80. Therefore, the number ofpatients, suffering from these two diseases, is increasing incessantlyas the prolonging of lifespans and the intensifying of population aging.AD, in particular, by far has affected more than 40 million patients,which would reach 150 million in 2050. For the United States alone, morethan 200 billion U.S. dollars are spent on AD patients caring per year,as twice as cancer, which makes it the most expensive disease of theworld. The number of PD patients of the world, according to aconservative estimation, has exceeded 10 million. However, the etiologyof these two diseases is still unknown. In terms of clinical treatment,although several drugs have been approved by the US FDA to treat mildand moderate AD or PD, these drugs are neurotransmitter regulating drugsthat can only temporarily improve the patient's cognitive or motorfunctions. The symptoms will rebound soon as soon as ceasing the drugs.Till now, no drug can terminate or reverse the pathological process ofthese two diseases. Therefore, it is extremely meaningful to develop newdrugs for the treatment of AD or PD.

The research finds that: The amyloid proteins in the brains of ADpatients are mainly β-amyloid (Aβ) protein and Tau protein, as well as asmall amount of α-synuclein (α-syn), and the initial site of onset isthe hippocampus that performs the functions of memory and learning andspatial orientation in the brain. The brain damage of PD patients startsfrom the substantia nigra, which is responsible for somatic motorfunction. The difference in the initial site of onset determinesdifferent symptoms of the patients with these two diseases. However,studies indicate that more than half of AD patients have dyskinesia inthe later stage, and most PD patients also share the same symptoms of ADpatients in the later stage. These phenomenons suggest that the twodiseases have intrinsic correlations in pathogenesis and diseaseprogression.

The formation of senile plaques in the brain is one of the basicpathological features of AD. As a main constituent substance in thesenile plaques, Aβ is a polypeptide consisting of 36-43 amino acids,which is a hydrolysis product of amyloid precursor protein (APP),wherein the content of Aβ(1-40) accounts for more than 90% of the totalamount of Aβ. The current study has clarified that although Aβ hasnormal physiological functions and can regulate acetylcholinergicsignaling between synapses by regulating the catalytic activity ofcholinesterase, but excessive aggregation and fibrosis of Aβ in thebrain can cause synapse dysfunction, and subsequent secondaryinflammatory response, leading to loss of neuronal function and neurondeath. Therefore, developing substances that can inhibit the aggregationand fibrosis of Aβ as well as block its neurotoxicity is one of theimportant approaches for the research and development of AD medication.

The pathological features of PD are mainly manifested as progressiveloss of dopamine (DA)-ergic neurons in the nigrostriatal system, alongwith the production of Lewy bodies. The Lewy bodies mainly comprisehollow radial amyloid fibers formed by the aggregation of denaturedα-syn. α-Syn is located at the presynaptic membrane terminal of neurons,and the natural state in the body is a soluble and unfolded state.Misfolding of α-Syn occurs under pathological conditions, generatesβ-sheet structures, which in turn are aggregated and fibrillated to formLewy body lesions. Research indicates that amyloidosis of α-syn plays akey role in the pathological process of the disease. Therefore,inhibiting α-syn aggregation and fibrosis has become one of theapproaches in the research and development of medication for PD'sprevention and treatment. On the other hand,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin. Itis not toxic per se, but after it enters the brain,1-methyl-4-phenylpyridine cation (MPP+) generated from its metabolismcan destroy DA-ergic neurons in the substantia nigra. At the same time,MPP+ can also interfere with NADH dehydrogenase, an important substancein the respiratory chain of mitochondrial metabolism, then causes celldeath and accumulation of free radicals. The mass death of DA-ergicneurons caused by this process severely affects the cerebral cortex'smotion control, resulting in similar symptoms of PD. Therefore, MPTP andMPP+ are widely used in the establishment of PD-related animal modelsand cell models as well as the research and development of PDmedications.

Gold nanoparticles are nanoscale gold particles (the diameter of thegold cores of the gold nanoparticles used in research is greater than 3nm in general). Because of the unique optical and electric properties,good biocompatibility as well as convenient surface modification, goldnanoparticles are widely used in biology and related medical fields suchas biosensors, medical imaging and tumor detection. Due to the chemicalinertness and large specific surface area and the ability to penetratethe blood-brain barrier at low concentrations, gold nanoparticles arealso used as drug carriers in the research of directional transport andcontrollable release of drugs, etc. In the recent years, research ismade on binding gold nanoparticles with specific ligands (such asheteropolyacids and specific sequence polypeptides) that inhibit theaggregation of fibrotic proteins, achieving certain effects in vitroprotein fibrosis inhibition experiments. (Y. H. Liao, Y. J. Chang, Y.Yoshiike, Y. C. Chang, Y. R. Chen, Small 2012, 8, 3631; Y. D. Alvarez,J. A. Fauerbach, J. V. Pellegrotti, T. M. Jovin, E. A. Jares-Erijman, F.D. Stefani, Nano Letters 2013, 13, 6156; S. Hsieh, C. W. Chang, H. H.Chou, Colloids and Surfaces B: Biointerfaces, 2013, 112, 525), but theresults of the cell model indicate that although there is a synergisticeffect on cell viability when gold nanoparticles (gold core size isabove 5 nm) are used together with compounds that have a protectiveeffect on fibrin damaged cells (N. Gao, H. Sun, K. Dong, J. Ren, X. Qu,Chemistry-A European Journal 2015, 21, 829), the effect is not obviouswhen they are used alone. AD experiments at the level of animal modelhave not yet been reported. Moreover, in these researches, goldnanoparticles were mainly used as drug carriers other than as activeingredients.

Gold clusters (AuCs) are ultrafine gold nanoparticles with a gold coreless than 3 nm in diameter. It contains only a few to hundreds of goldatoms, causing the face-centered cubic packing structure of the goldatoms in the conventional gold nanoparticles to collapse and the energylevel to split, thus showing molecule-like properties that arecompletely different from the conventional gold nanoparticles of above 3nm: On the one hand, due to energy level splitting, AuCs do not possessthe surface plasmon effect and derived optical properties ofconventional gold nanoparticles, but exhibit excellent fluorescenceemission properties similar to semiconductor quantum dots. On the otherhand, in the ultraviolet-visible absorption spectrum of AuCs, theplasmon resonance peak at 520±20 nm disappears, while one or more newabsorption peaks appear above 560 nm, and such absorption peaks cannotbe observed in conventional gold nanoparticles. Therefore, thedisappearance of the plasmon resonance absorption peak (520±20 nm) andthe appearance of the new absorption peaks above 560 nm in theUV-visible absorption spectrum are important indicators for judgingwhether AuCs are successfully prepared (H. F. Qian, M. Z. Zhu, Z. K. Wu,R. C. Jin, Accounts of Chemical Research 2012, 45, 1470). AuCs also havemagnetic, electrical and catalytic properties and photothermal effectsthat are significantly different from those of conventional goldnanoparticles, so they have broad application prospects in the fields ofsingle-molecule optoelectronics, molecular catalysis, and photothermalconversion.

In addition, AuCs have also been used in the fields of bioprobes andmedical imaging due to their excellent fluorescence emission properties.For example, Sandeep Verma team uses purine-modified AuCs as greenfluorescent probes for nucleus imaging, (J. R. Wallbank, D. Ghazaryan,A. Misra, Y. Cao, J. S. Tu, B. A. Piot, M. Potemski, S. Wiedmann, U.Zeitler, T. L. M. Lane, S. V. Morozov, M. T. Greenaway, L. Evaes, A. K.Geim, V. I. Falko, K S. Novoselov, A. Mishchenko, ACS Applied Materials& Interfaces 2014, 6, 2185). This type of literatures utilizes thefluorescence properties of AuCs and does not involve the medicinalactivity of AuCs themselves.

SUMMARY OF THE INVENTION

The objective of the present invention is to tackle the technicaldefects in the prior art. In the first aspect, the present inventionprovides an AuCs-containing substance that has medicinal activity andcomprises AuCs and ligand Y coating AuCs externally.

The gold core diameter of the AuCs is smaller than 3 nm, preferably0.5-2.6 nm.

The ligand Y includes, but not limited to, one or more of L(D)-cysteineand its derivatives, cysteine-containing oligopeptides and theirderivatives, and other thiol-containing compounds.

The L(D)-cysteine and its derivatives are preferably L(D)-cysteine,N-isobutyryl-L(D)-cysteine (L(D)-NIBC) or N-acetyl-L(D)-cysteine(L(D)-NAC).

The cysteine-containing oligopeptides and their derivatives arepreferably cysteine-containing dipeptides, cysteine-containingtripeptides or cysteine-containing tetrapeptides.

The cysteine-containing dipeptides are preferably L-cysteine-L-argininedipeptide (CR), L-arginine-L-cysteine dipeptide (RC),L-histidine-L-cysteine dipeptide (HC) or L-cysteine-L-histidinedipeptide (CH).

The cysteine-containing tripeptides are preferablyglycine-L-cysteine-L-arginine tripeptide (GCR),L-proline-L-cysteine-L-arginine tripeptide (PCR),L-lysine-L-cysteine-L-proline tripeptide (KCP) or L-glutathione (GSH).

The cysteine-containing tetrapeptides are preferablyglycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR) orglycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR).

Other thiol-containing compounds are preferably1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycollic acid,mercaptoethanol, thiophenol, D-3-trolovol,N-(2-mercaptopropionyl)-glycine or dodecyl mercaptan.

The substance is powder or floc.

In the second aspect, the present invention provides a method forpreparing the AuCs-containing substance, said method comprising thefollowing steps:

(1) Dissolving HAuCl₄ in one of methanol, water, ethanol, n-propanol andethyl acetate to get a solution A in which the concentration of HAuCl₄is 0.01˜0.03M;

(2) Dissolving ligand Y in a solvent to get a solution B in which theconcentration of ligand Y is 0.01˜0.18M;

(3) Mixing the solution A in step (1) with the solution B in step (2) ata mole ratio between HAuCl₄ and ligand Y 1: (0.01˜100) (preferably 1:(0.1-10), more preferably 1: (1-10)), stirring them in an ice bath for0.1˜48 h (preferably 0.1-24 h, more preferably 0.5-2 h), adding0.025˜0.8M NaBH₄ solution (preferably water solution of NaBH₄, ethanolsolution of NaBH₄ or methanol solution of NaBH₄), and then continuing tostir in an ice water bath for 0.1˜12 h (preferably 0.1-2 h, morepreferably 1-2 h), at a mole ratio between NaBH₄ and ligand Y 1:(0.01˜00) (preferably 1: (0.1-8), more preferably 1: (1-8));

(4) Centrifuging the reaction solution in step (3) at 8000˜17500 r/minfor 10˜100 min to obtain AuCs precipitate in different average particlesizes; preferably, using MWCO 3K˜30K ultrafiltration tubes to centrifugethe reaction solution in step (3) at 8000˜17500 r/min by gradient for10˜100 min to obtain AuCs in different average particle sizes;

(5) Dissolving the AuCs precipitate in different average particle sizesobtained in step (4) in water, putting it in a dialysis bag anddialyzing it in water at room temperature for 1˜7 days;

(6) Freeze-drying the AuCs solution in the dialysis bag for 12˜24 h toobtain the AuCs-containing substance.

The solvent in step (2) is one or more of methanol, ethyl acetate,water, ethanol, n-propanol, pentane, formic acid, acetic acid, diethylether, acetone, anisole, 1-propanol, 2-propanol, 1-butanol, 2-butanol,pentanol, butyl acetate, tributyl methyl ether, isopropyl acetate,dimethyl sulfoxide, ethyl formate, isobutyl acetate, methyl acetate,2-methyl1-propanol and propyl acetate.

In the third aspect, the present invention provides the application ofthe AuCs-containing substance in near-infrared fluorescent probes in thefields of catalyst preparation or molecular catalysis, chiralrecognition, molecular detection, biomedical detection and imaging.

In the fourth aspect, the present invention provides the application ofthe AuCs-containing substance in the preparation of drugs for thedisease associated with the aggregation and fibrosis of Aβ and for thedisease associated with the aggregation and fibrosis of α-syn.

In the fifth aspect, the present invention provides the application ofthe AuCs-containing substance in the preparation of drugs for preventionand treatment of AD.

In the sixth aspect, the present invention provides the application ofthe AuCs-containing substance in the preparation of drugs for preventionand treatment of PD.

In the seventh aspect, the present invention provides the application ofAuCs in the preparation of drugs for the disease associated with theaggregation and fibrosis of Aβ.

The disease associated with the aggregation and fibrosis of Aβ is AD.

The AuCs are modified with L-glutathione (GSH), N-acetyl-L(D)-cysteine(L(D)-NAC), N-isobutyryl-L(D)-cysteine (L(D)-NIBC),L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide(RC), 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (Cap) orL(D)-cysteine (L(D)-Cys).

In the eighth aspect, the present invention provides the application ofAuCs in the preparation of medication for the disease associated withthe α-syn aggregation and fibrosis.

The disease associated with the α-syn aggregation and fibrosis is PD.

The AuCs are modified with L-glutathione (GSH), N-acetyl-L(D)-cysteine(L(D)-NAC), N-isobutyryl-L(D)-cysteine (L(D)-NIBC),L-cysteine-L-arginine dipeptide (CR), L-arginine-L-cysteine dipeptide(RC), 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (Cap) orL(D)-cysteine (L(D)-Cys).

The AuCs-containing substance provided by the present invention showsexcellent effect on inhibiting Aβ and α-syn aggregation in the in vitroexperiment for Aβ and α-syn aggregation inhibition, and shows excellenteffect on improving cell viability in Aβ induced cell AD model and MPP⁺induced cell PD model experiments. In the transgenic mouse model of AD,the AuCs-containing substance can significantly improve the cognitivebehavioral ability of sick mice, and plays a significant role ininhibiting the formation of Aβ (1-40) and Aβ (1-42) plaques in thehippocampus and cerebral cortex of mice. In the MPTP-induced PD mousemodel, the AuCs-containing substance can significantly ameliorate andcorrect the dyskinetic disorder of MPTP lesion model mice, improve themotor abilities of sick mice, and substantially inhibit MPTP-inducedspecific apoptosis of DA-ergic neurons of substantia nigra and striatumof mice. And it also has good biosafety at the cell level and animallevel. The above results indicate that the AuCs-containing substanceprovided by the present invention not only affects the aggregation andfibrosis of fibrotic proteins, but also influences the process ofneurodegenerative diseases at deeper levels such as signaling functionsrelated to energy metabolism and neurotransmitter metabolism of nervecells. Therefore, the AuCs-containing substance provided by the presentinvention is important for the research and development of newmedication for neurodegenerative diseases such as AD and/or PD.

On the other hand, since ligand molecules did not show any inhibitoryeffect in the kinetic experiment for in vitro inhibition of Aβaggregation, and Aβ-lesioned AD cell model test, or any increase of cellviability in Aβ lesioned cell AD model and MPP⁺-lesioned PD cell model,this suggests that the efficacy to AD and PD comes from AuCs other thanligands. Based on the medicinal activity of AuCs itself, development ofcompetitive new medication is expected.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows ultraviolet-visible (UV) spectrums, transmission electronmicroscope (TEM) images and particle size distribution diagrams ofligand L-NIBC-modified gold nanoparticles with different particle sizes.

FIG. 2 shows ultraviolet visible spectrums, TEM images and particle sizedistribution diagrams of ligand L-NIBC-modified AuCs with differentparticle sizes.

FIG. 3 shows infrared spectrums of ligand L-NIBC-modified AuCs withdifferent particle sizes.

FIG. 4 shows AFM topographies after Aβ (1-40) and ligand L-NIBC-modifiedgold nanoparticles or AuCs are jointly incubated for 48 h.

FIG. 5 shows kinetic curves of Aβ fibrosis of ligand L-NIBC-modifiedgold nanoparticles and AuCs of different particle sizes and differentconcentrations.

FIG. 6 shows diagrams showing the effects of ligand L-NIBC-modified goldnanoparticles or AuCs of different particle sizes and differentconcentrations on the cell viability of Aβ-induced AD cell model.

FIG. 7 shows UV, infrared, TEM and particle size distribution diagramsof AuCs modified with ligand CR (CR-AuCs).

FIG. 8 shows UV, infrared, TEM and particle size distribution diagramsof AuCs modified with ligand RC (RC-AuCs).

FIG. 9 shows UV, infrared, TEM and particle size distribution diagramsof AuCs modified with ligand1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (i.e., Cap).

FIG. 10 shows UV, infrared, TEM and particle size distribution diagramsof AuCs modified with ligand GSH (GSH-AuCs).

FIG. 11 shows UV, infrared, TEM and particle size distribution diagramsof AuCs modified with ligand D-NIBC (D-NIBC-AuCs).

FIG. 12 shows curves of the inhibitory effect of AuCs modified withdifferent ligands on the aggregation and fibrosis of Aβ (1-40).

FIG. 13 shows a schematic diagram of a water maze experiment device inEmbodiment 5.

FIG. 14 shows diagrams showing the effect of an AuCs-containingsubstance on the cognitive behavior (day 150 of administration) ofAPP/PS1 double transgenic C57BL/6 mouse model.

FIG. 15 shows diagrams showing the effect of an AuCs-containingsubstance on the expression of Aβ (1-40) in the hippocampus and cerebralcortex of mice in APP/PS1 double transgenic C57BL/6 mouse model (day 100of administration).

FIG. 16 shows diagrams showing the effect of an AuCs-containingsubstance on the expression of Aβ (1-42) in the hippocampus and cerebralcortex of mice in APP/PS1 double transgenic C57BL/6 mouse model (day 100of administration).

FIG. 17 shows diagrams showing the effect of an AuCs-containingsubstance on the expression of Aβ (1-40) in the hippocampus and cerebralcortex of mice in APP/PS1 double transgenic C57BL/6 mouse model (day 150of administration).

FIG. 18 shows diagrams showing the effect of an AuCs-containingsubstance on the expression of Aβ (1-42) in the hippocampus and cerebralcortex of mice in APP/PS1 double transgenic C57BL/6 mouse model (day 150of administration).

FIG. 19 is a diagram showing the effect of a substance containing AuCson α-syn fibrosis kinetics.

FIG. 20 shows diagrams showing the effect of an AuCs-containingsubstance on the cell viability of MPP⁺-lesioned PD cell (SH-sy5y)model.

FIG. 21 shows diagrams showing the effect of an AuCs-containingsubstance on cell apoptosis of MPP⁺-induced PD cell (PC12) model.

FIG. 22 shows diagrams showing the effect of an AuCs-containingsubstance on the locomotor activity of MPTP-lesioned model mice.

FIG. 23 shows diagrams showing the effect of an AuCs-containingsubstance on the swimming ability of the MPTP-lesioned model mice.

FIG. 24 shows diagrams showing the effect of an AuCs-containingsubstance on the rotarod behavior of the MPTP-lesioned model mice.

FIG. 25 shows diagrams showing the effect of an AuCs-containingsubstance on DA-ergic neurons in substantia nigra and striatium of theMPTP-lesioned model mice.

FIG. 26 shows diagrams showing the effect of an AuCs-containingsubstance of different particle sizes and different concentrations onSH-sy5y neuroblastoma cell viability.

DETAILED DESCRIPTION OF THE EMBODIMENTS

When studying the effect of gold nanoparticles with certain ligands onAβ aggregation, the inventors found that when the gold core diameter ofgold nanoparticles was changed from large to small, the promoting effectof gold nanoparticles modified with the same ligand on their surfaces onAβ aggregation was converted into inbibitory one; when the particle sizewas small enough to become AuCs, complete inhibition of Aβ aggregationcould be achieved. In addition, it was also found that AuCs also had acomplete inhibitory effect on α-syn. In this effect, it is AuCsthemselves other than ligands to play an inhibitory role.

Generally, the gold core diameter of the gold nanoparticles used in theresearch is greater than 3 nm, and when the diameter of the gold core issmaller than 3 nm, they are called AuCs. The disappearance of plasmonresonance absorption peak (520±20 nm) and the appearance of newabsorption peaks above 560 nm in UV-visible absorption spectrum indicatethat the AuCs are prepared successfully. Without ligands, AuCs cannotexist stably in a solution. It combines with thiol-containing ligand toform ligand-modified AuCs (or called AuCs) via Au-S bond.

The existing ligand-modified AuCs disclosed in the literature includeAuCs modified with L-glutathione (GSH), N-acetyl-L(D)-cysteine(L(D)-NAC), N-isobutyryl-L(D)-cysteine (L(D)-NIBC), etc. The preparationprocess is shown in the literatures (H. F. Qian, M. Z. Zhu, Z. K. Wu, RC. Jin, Accounts of Chemical Research 2012, 45, 1470; C. Gautier, T.Burgi, Journal of the American Chemical Society 2006, 128, 11079); theyare mainly applied in the fields of catalysis, chiral recognition,molecular detection, biosensing, drug delivery and bioimaging (G. Li, R.C. Jin, Accounts of Chemical Research 2013, 46, 1749; H. F. Qian, M. Z.Thu, Z. K. Wu, R. C. Jin, Accounts of Chemical Research 2012, 45, 1470;J. F. Parker, C. A. Fields-Zinna, R. W. Murray, Accounts of ChemicalResearch 2010, 43, 1289; S. H. Yau, O. Varnayski, T. Goodson, Accountsof Chemical Research 2013, 46, 1506).

The present invention investigated the effects of AuCs on AD and/or PD,at least including: firstly, AuCs of different sizes containingdifferent ligands (the ligands not having inhibitory effect on Aβaggregation) were used as research objects. Through research at threelevels of experiments, including in vitro experiments for inhibition ofAβ aggregation and -syn aggregation, Aβ induced AD cell model and MPP⁺induced PD cell model experiments, and AD transgenic mouse model andMPTP induced PD mouse model experiments, and in consideration of AuCscytotoxicity, acute toxicity experiment in mice, in vivo distributionexperiment in mice, etc., ligand-modified AuCs were provided, theirapplication in the preparation of drugs treating AD and PD was found,and the results were compared with the experimental results of goldnanoparticles, indicating that gold nanoparticles with a diameter ofgreater than 3 nm do not have a desirable effect for this purpose, andcannot be used to prepare drugs treating AD or PD, while ligand-modifiedAuCs can be used to prepare drugs treating AD and/or PD.

Hereunder the present invention will be further detailed in embodiments,but those embodiments should not be understood as constituting anylimitation to the present invention.

The purity of the raw materials used in the following embodiments shallbe chemical purity or higher. They all may be purchased from the market.

Embodiment 1: Prepare Ligand-Modified AuCs

This embodiment discloses a method for preparing ligand-modified AuCs,said method comprising the following steps:

(1) Dissolving HAuCl₄ in one of methanol, water, ethanol, n-propanol andethyl acetate to get a solution A in which the concentration of HAuCl₄is 0.01˜0.03M;

(2) Dissolving ligand Y in a solvent to get a solution B in which theconcentration of ligand Y is 0.01˜0.18M; ligand Y includes, but notlimited to, L(D)-cysteine and other cysteine derivatives such asN-isobutyryl-L-cysteine (L-NIBC), N-isobutyryl-D-cysteine (D-NIBC),N-acetyl-L-cysteine and N-acetyl-D-cysteine, cysteine-containingoligopeptides and their derivatives including, but not limited to,dipeptides, tripeptide, tetrapeptide and other peptides containingcysteine, such as L-cysteine-L-arginine dipeptide (CR),L-arginine-L-cysteine dipeptide (RC), L-cysteine L-histidine (CH),glycine-L-cysteine-L-arginine tripeptide (GCR),L-proline-L-cysteine-L-arginine tripeptide (PCR), L-glutathione (GSH),glycine-L-serine-L-cysteine-L-arginine tetrapeptide (GSCR) andglycine-L-cysteine-L-serine-L-arginine tetrapeptide (GCSR), and otherthiol-containing compounds, such as one or more of1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline, thioglycollic acid,mercaptoethanol, thiophenol, D-3-trolovol and dodecyl mercaptan; thesolvent is one or more of methanol, ethyl acetate, water, ethanol,n-propanol, pentane, formic acid, acetic acid, diethyl ether, acetone,anisole, 1-propanol, 2-propanol, 1-butanol, 2-butanol, pentanol, butylacetate, tributyl methyl ether, isopropyl acetate, dimethyl sulfoxide,ethyl formate, isobutyl acetate, methyl acetate, 2-methyl-1-propanol andpropyl acetate;

(3) Mixing solution A and solution B so that the mole ratio betweenHAuCl₄ and ligand Y is 1: (0.01˜100), stirring them in an ice bath for0.1˜48 h, adding 0.025˜0.8M NaBH₄ water, ethanol or methanol solution,continuing to stir in an ice water bath and react for 0.1˜12 h. The moleratio between NaBH₄ and ligand Y is 1: (0.01˜100);

(4) Using MWCO 3K˜30K ultrafiltration tubes to centrifuge the reactionsolution at 8000˜17500 r/min by gradient for 10˜100 min after thereaction ends to obtain ligand-modified AuCs precipitate in differentaverage particle sizes (the specific gradient centrifugation is asdescribed in (4) of Embodiment 2. The aperture of the filtrationmembranes for ultrafiltration tubes of different MWCOs directly decidesthe size of AuCs that can pass the membranes). This step may be omitted.In other words, after step (3) is completed, step (5) is starteddirectly to obtain mixed AuCs in different sizes;

(5) Dissolving the AuCs precipitate in different average particle sizesobtained in step (4) in water, putting it in a dialysis bag anddialyzing it in water at room temperature for 1˜7 days;

(6) Freeze-drying AuCs for 12˜24 h after dialysis to obtain a powdery orflocculant substance, i.e., ligand-modified AuCs.

As detected (the specific detection method is shown in Embodiment 2),the particle size of the powdery or flocculant substance obtained by theforegoing method is smaller than 3 nm (distributed in 0.5-2.6 nm ingeneral). The UV-visible absorption spectrum has one or more absorptionpeaks above 560 nm, and no obvious absorption peak at 520 nm. It isdetermined that the obtained powder or floc is AuCs.

Embodiment 2: Preparation and Confirmation of AuCs Modified withDifferent Ligands

Taking ligand L-NIBC for example, the preparation and confirmation ofAuCs modified with ligand L-NIBC are detailed.

(1) Weigh 1.00 g of HAuCl₄ and dissolve it in 100 mL of methanol toobtain a 0.03M solution A;

(2) Weigh 0.57 g of L-NIBC and dissolve it in 100 mL of glacial aceticacid (acetic acid) to obtain a 0.03M solution B;

(3) Measure 1 mL of solution A, mix it with 0.5 mL, 1 mL, 2 mL, 3 mL, 4mL, 5 mL of solution B respectively (i.e. the mole ratio between HAuCl₄and L-NIBC is 1:0.5, 1:1, 1:2, 1:3, 1:4, 1:5 respectively), react in anice bath under stirring for 2 h, quickly add 1 mL of freshly prepared0.03M (prepared by weighing 11.3 mg of NaBH₄ and dissolving it in 10 mLof ethanol) NaBH₄ ethanol solution when the solution turns colorlessfrom bright yellow, continue the reaction for 30 min after the solutionturns dark brown, and add 10 mL of acetone to terminate the reaction.

(4) After the reaction, the reaction solution is subjected to gradientcentrifugation to obtain L-NIBC modified AuCs powder with differentparticle sizes. Specific method: After the reaction is completed, thereaction solution is transferred to an ultrafiltration tube with MWCO of30K and a volume of 50 mL, and centrifuged at 10000 r/min for 20 min,and the retentate in the inner tube is dissolved in ultrapure water toobtain powder with a particle size of about 2.6 nm. Then, the mixedsolution in the outer tube is transferred to an ultrafiltration tubewith a volume of 50 mL and MWCO of 10K, and centrifuged at 13,000 r/minfor 30 min. The retentate in the inner tube is dissolved in ultrapurewater to obtain powder with a particle size of about 1.8 nm. Then themixed solution in the outer tube is transferred to an ultrafiltrationtube with a volume of 50 mL and MWCO of 3K, and centrifuged at 17,500r/min for 40 min. The retentate in the inner tube is dissolved inultrapure water to obtain powder with a particle size of about 1.1 nm.

(5) Precipitate the powder in three different particle sizes obtained bygradient centrifugation, remove the solvent respectively, blow the crudeproduct dry with N₂, dissolve it in 5 mL of ultrapure water, put it in adialysis bag (MWCO is 3KDa), put it in 2 L of ultrapure water, changewater every other day, dialyze it for 7 days, freeze-dry it and keep itfor future use.

Characterization experiment was conducted for the powder obtained above(ligand L-NIBC modified AuCs). Meanwhile, ligand L-NIBC modified goldnanoparticles are used as control. The method for preparing goldnanoparticles with ligand being L-NIBC refers to the reference (W. Yan,L. Xu, C. Xu, W Ma, H. Kuang, L. Wang and N. A. Kotov, Journal of theAmerican Chemical Society 2012, 134, 15114; X. Yuan, B. Zhang, Z. Luo,Q. Yao, D. T. Leong, N. Yan and J. Xie, Angewandte Chemie InternationalEdition 2014, 53, 4623).

1. Observe the Morphology by Transmission Electron Microscope (TEM)

The test powders (L-NIBC modified AuCs sample prepared in Embodiment 2and L-NIBC modified gold nanoparticle sample) was dissolved in ultrapurewater to 2 mg/L as samples, and then test samples were prepared byhanging drop method. The specific method: 5 μL of the samples weredripped on an ultrathin carbon film, volatized naturally till the waterdrop disappeared, and then observe the morphology of the samples byJEM-2100F STEM/EDS field emission high-resolution TEM.

The four TEM images of ligand L-NIBC modified gold nanoparticles areshown in panels B, E, H, and K of FIG. 1; the three TEM images of ligandL-NIBC modified AuCs are shown in panels B, E, and H of FIG. 2.

The images in FIG. 2 indicate that the L-NIBC-modified AuCs samples havea uniform particle size and good dispersibility, and the averagediameter of L-NIBC-modified AuCs (refer to the diameter of gold core) is1.1 nm, 1.8 nm and 2.6 nm respectively, in good accordance with theresults in panels C, F and I of FIG. 2. In comparison, ligand L-NIBCmodified gold nanoparticle samples have a larger particle size. Theiraverage diameter (refer to the diameter of gold core) is 3.6 nm, 6.0 nm,10.1 nm and 18.2 nm respectively, in good accordance with the results inpanels C, F, I and L of FIG. 1.

2. Ultraviolet (UV)-Visible (Vis) Absorption Spectrum

The test powder was dissolved in ultrapure water till the concentrationwas 10 mg·L⁻¹, and was measured by UV-vis absorption spectrum at roomtemperature. The scanning range was 190-1100 nm, the sample cell was astandard quartz cuvette with an optical path of 1 cm, and the referencecell was filled with ultrapure water.

The UV-vis absorption spectra of the four ligand L-NIBC-modified goldnanoparticle samples with different sizes are shown in panels A, D, Gand J of FIG. 1, and the statistical distribution of particle size isshown in panels C, F, I and L of FIG. 1; the UV-vis absorption spectraof three ligand L-NIBC modified AuCs samples with different sizes areshown in panels A, D and G of FIG. 2, and the statistical distributionof particle size is shown in panels C, F and I of FIG. 2.

FIG. 1 indicates that due to the surface plasmon effect, ligand L-NIBCmodified gold nanoparticles had an absorption peak at about 520 nm. Theposition of the absorption peak is relevant with particle size. When theparticle size is 3.6 nm, the UV absorption peak appears at 516 nm; whenthe particle size is 6.0 nm, the UV absorption peak appears at 517 nm;when the particle size is 10.1 nm, the UV absorption peak appears at 520nm, and when the particle size is 18.2 nm, the absorption peak appearsat 523 nm. None of the four samples has any absorption peak above 560nm.

FIG. 2 indicates that in the UV absorption spectra of three ligandL-NIBC-modified AuCs samples with different particle sizes in Embodiment2, the surface plasmon effect absorption peak at 520 nm disappeared, andtwo obvious absorption peaks appeared above 560 nm and the positions ofthe absorption peaks varied slightly with the particle sizes of AuCs.This is because AuCs exhibit molecule-like properties due to thecollapse of the face-centered cubic structure, which leads to thediscontinuity of the density of states of AuCs, the energy levelsplitting, the disappearance of plasmon resonance effect and theappearance of a new absorption peak in the long-wave direction. It couldbe concluded that the three powder samples in different particle sizesobtained in Embodiment 2 are all ligand-modified AuCs.

3. Fourier Transform Infrared Spectroscopy

Infrared spectra were measured on a VERTEX80V Fourier transform infraredspectrometer manufactured by Bruker in a solid powder high vacuum totalreflection mode. The scanning range is 4000-400 cm⁻¹ and the number ofscans is 64. Taking the L-NIBC modified AuCs samples prepared inEmbodiment 2 for example, the test samples were L-NIBC modified AuCs drypowder with three different particle sizes and the control sample waspure L-NIBC powder. The results are shown in FIG. 3.

FIG. 3 shows the infrared spectrum of L-NIBC modified AuCs withdifferent particle sizes. Compared with pure L-NIBC (the curve at thetop), the S-H stretching vibrations of L-NIBC modified AuCs withdifferent particle sizes all disappeared completely at 2500-2600 cm⁻¹,while other characteristic peaks of L-NIBC were still observed, provingthat L-NIBC molecules were successfully anchored to the surface of AuCsvia Au-S bond. The figure also shows that the infrared spectrum of theligand-modified AuCs is irrelevant with its size.

AuCs modified by other ligand Y were prepared by a method similar to theabove method, except that the solvent of solution B, the feed ratiobetween HAuCl₄ and ligand Y, the reaction time and the amount of NaBH₄added were slightly adjusted. For example: when L-cysteine, D-cysteine,N-isobutyryl-L-cysteine (L-NIBC) or N-isobutyryl-D-cysteine (D-NIBC) isused as ligand Y, acetic acid is selected as the solvent; when dipeptideCR, dipeptide RC or 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline isused as ligand Y, water is selected as the solvent, and so on and soforth; other steps are similar, so they won't be described in detailshere.

The present invention prepared and obtained a series of ligand-modifiedAuCs by the foregoing method. The ligands and the parameters of thepreparation process are shown in Table 1.

TABLE 1 Preparation parameters of AuCs modified with different ligandsin the present invention Parameter Feed ratio Time of reaction Moleratio Time of reaction between in an ice bath between in an ice bathSolvent used HAuCl₄ under stirring before HAuCl₄ under stirring afterEmbodiments Ligand Y for solution B and Y addition of NaBH₄ and NaBH₄addition of NaBH₄ 1 L-cysteine Acetic acid 1:3 2 h 1:2 0.5 h 2D-cysteine Acetic acid 1:3 2 h 1:2 0.5 h 3 N-acetyl-L-cysteine Ethanol1:4 1 h 1:1 0.5 h 4 N-acetyl-D-cysteine Ethanol 1:4 1 h 1:1 0.5 h 5L-NIBC Water 1:4 0.5 h 1:2 0.5 h 6 D-NIBC Water 1:4 0.5 h 1:2 0.5 h 7Other cysteine derivatives Soluble solvent 1:(0.1~100) 0.5 h~24 h1:(0.1~100) 0.1~24 h 8 CR Water 1:4 22 h 2:1 0.5 h 9 RC Water 1:4 20 h2:1 0.5 h 10 HC Water 1:3 12 h 1:2 2 h 11 CH Ethanol 1:4 16 h 1:3 3 h 12GSH Water 1:2 12 h 1:1 3 h 13 KCP Water 1:3 15 h 1:2 1 h 14 PCR Water1:4 16 h 1:3 2 h 15 GSCR Water 1:4 16 h 1:3 1.5 h 16 GCSR Water 1:3 12 h1:2 2 h 17 Other oligopeptides Soluble solvent 1:(0.1~100) 0.5 h~24 h1:(0.1~100) 0.1~24 h containing cysteine 18 1-[(2S)-2-methyl-3-thiol-Water 1:8 2 h 1:7 1 h 1-oxopropyl]-L-proline 19 Mercaptoethanol Ethanol1:2 2 h 1:1 1 h 20 Thioglycollic acid Acetic acid 1:2 2 h 1:1 1 h 21Thiophenol Ethanol 1:5 5 h 1:1 1 h 22 D-3-trolovol Water 1:2 2 h 1:1 1 h23 N-(2-mercaptopropionyl)- Water 1:2 2 h 1:1 1 h glycine 24 Dodecylmercaptan Methanol 1:5 5 h 1:1 1 h 25 Other compounds Soluble solvent1:(0.01~100) 0.5 h~24 h 1:(0.1~100) 0.1~24 h containing thiol

The samples in embodiments listed in Table 1 are confirmed by theforegoing method. FIG. 7-FIG. 11 are UV spectra (panel A in FIG. 7-FIG.11), infrared spectra (panel B in FIG. 7-FIG. 11), transmission electronmicroscope (TEM) images (panel C in FIG. 7-FIG. 11) and particle sizedistribution (panel D in FIG. 7-FIG. 11) of AuCs modified with ligandCR, RC, 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (abbreviation:Cap), GSH and D-NIBC.

The results indicate that the diameters of AuCs modified with differentligands obtained from Table 1 are all smaller than 3 nm. Ultravioletspectra also show disappearance of peak at 520±20 nm, and appearance ofabsorption peak above 560 nm. Only the position of this absorption peakvaries slightly with ligand and particle size. Meanwhile, Fouriertransform infrared spectrum also shows disappearance of ligand thiolinfrared absorption peak (between the dotted lines in panel B of FIG.7-FIG. 11), while other infrared characteristic peaks are all retained,suggesting that all ligand molecules have been successfully anchored tothe surface of AuCs, and the present invention has successfully obtainedAuCs modified with the ligands listed in Table 1.

Embodiment 3: In Vitro Aβ Aggregation Kinetic Experiment

This embodiment validated the functions of ligand-modified AuCs by invitro experiment of Aβ aggregation kinetics, and compared the effects onAβ aggregation kinetics with ligand-modified gold nanoparticles andindependent use of ligand molecules to prove that the function is fromAuCs other than ligand. The experiment used ThT fluorescent labelingmethod to characterize the kinetics of Aβ(1-40) aggregation andfibrosis.

Thioflavin T (abbreviation: ThT) is a dye specifically for dyeingamyloid fibers. When it is incubated together with monomers ofpolypeptides or proteins, its fluorescence does not changesubstantially. When it encounters amyloid polypeptides or proteins witha fiber structure, it will immediately couple with the amyloidpolypeptides or proteins and its fluorescence intensity will increaseexponentially. Just because of this property, ThT is widely used as amarker to monitor amyloidosis of peptides or proteins. The fibrosisprocess of Aβ(1-40) is also a nucleation-controlled polymerizationprocess. Therefore, the growth curve of Aβ(1-40) fiber measured by ThTfluorescent labeling method is mainly divided into three stages: Initialstage, growth stage and platform stage. The initial stage is mainly astage when Aβ(1-40) undergoes conformational transition to formmisfolding and then aggregates and nucleates. The growth stage is astage when Aβ(1-40) monomers are accumulated onto the cores of oligomersalong the axial direction to form fibers and grow rapidly. The platformstage is a stage when all Aβ(1-40) molecules form mature long fibers,i.e., a stage when the fibers no longer grow. ThT fluorescent labelingmethod can conveniently monitor the kinetics process of fibroticaggregation of Aβ(1-40) molecules.

1) Pretreatment of Aβ(1-40) Monomers

Freeze-dried powder of amyloid polypeptide Aβ(1-40) (Invitrogen Corp.)was dissolved in hexafluoroisopropanol (HFIP) to obtain a 1 g/L Aβ(1-40)solution, and the solution was inculated at room temperature for 2-4 hafter sealing, then blowed to dry HFIP (for about 1 h) with high-puritynitrogen (N2, 99.9%) at an appropriate flow rate in a fume hood. Lastlythe dried Aβ(1-40) was dissolved in 200 μL of DMSO, and after sealing,the solution was kept in a refrigerator at −20° C. for no more than oneweek for future use. Before use, the DMSO solution of the amyloidpolypeptide was diluted with profuse phosphate buffer solution (PBS, 10mM, pH=7.4) till the concentration of Aβ(1-40) reached 20 μM to obtainan Aβ(1-40) PBS solution. All the Aβ(1-40) PBS solutions for theexperiments were prepared freshly.

2) Sample Preparation and Detection

Ligand-modified AuCs and gold nanoparticles were added to 20 μM Aβ(1-40)PBS respectively to form samples of AuCs modified with different ligandsat different concentrations and different particle sizes, and samples ofgold nanoparticles modified with different ligands correspondingly. Thesamples were incubated continuously in a 96-well plate at 37° C. by ThTfluorescent labeling method, and monitored the fluorescence intensity bymicroplate reader once every 10 minutes. The kinetic process of Aβ(1-40)aggregation was characterized through the change of fluorescenceintensity of ThT.

Three sizes of L-NIBC-modified AuCs with particle sizes of 2.6 nm, 1.8nm and 1.1 nm respectively prepared in Embodiment 2 were used asexperiment groups. Four sizes of L-NIBC-modified gold nanoparticles withparticle sizes of 18.2 nm, 10.1 nm, 6.0 nm and 3.6 nm respectively, andL-NIBC molecules uncombined with AuCs or gold nanoparticles were used ascontrol groups. Every size of AuCs or gold nanoparticles were in sixconcentrations respectively, which were: 0 ppm (not containing AuCs,gold nanoparticles or L-NIBC, as blank control), 0.1 ppm, 1.0 ppm, 5.0ppm, 10.0 ppm and 20.0 ppm respectively. L-NIBC molecules in twoconcentrations were used, which were: 1.0 ppm and 10.0 ppm respectively.

The results are shown in FIG. 4 and FIG. 5.

FIG. 4 shows AFM topographies of Aβ(1-40) after coincubating with eachexperiment group and control group for 48 h. Panel A is the AFMtopography after Aβ(1-40) was incubated alone for 48 h. Panel B is anAFM topography after Aβ(1-40) was coincubated with L-NIBC for 48 h.Panel C and panel D are AFM topographies after Aβ(1-40) was coincubatedwith gold nanoparticles with an average particle size of 6.0 nm and 3.6nm respectively (modified with L-NIBC) for 48 h. And panel E is an AFMtopography after Aβ(1-40) was coincubated with AuCs in an averageparticle size of 1.8 nm (modified with L-NIBC) for 48 h.

In FIG. 5, the amyloidosis kinetics curve of Aβ(1-40) in differentconcentrations of L-NIBC is shown in panel A. The amyloidosis kineticscurves of Aβ(1-40) in different concentrations of gold nanoparticleswith sizes of 18.2 nm, 10.1 nm, 6.0 nm and 3.6 nm respectively are shownin panel B-E. The amyloidosis kinetics curves of Aβ(1-40) in differentconcentrations of AuCs with sizes of 2.6 nm, 1.8 nm and 1.1 nmrespectively are shown in panel F-H. The amyloidosis kinetics curves ofAβ in panels A-H are curves when Aβ(1-40) was coincubated goldnanoparticles or AuCs in different concentrations, □ represented 0 ppm(i.e., no gold nanoparticles and AuCs), ∘ represented 0.1 ppm, Δrepresented 1 ppm, represented 5 ppm, represented 10 ppm, ⋆ represented20 ppm.

It could be seen from FIG. 4 that as control, Aβ fibers was filled inpanel A; the same as panel B; though fibers reduced to some extent, longfibers could still be seen in panel C; though there was no long fibers,many Aβ short fibers still existed in panel D. It was indicated thatL-NIBC had no obvious effect on the formation of Aβ(1-40) fibers. Theaddition of L-NIBC-modified small-size gold nanoparticles could delaythe amyloidosis process of Aβ(1-40), but not inhibit completely becauseshort fibers would continue to grow into long fibers after more time. Ithas neither long fibers nor short fibers in panel E of FIG. 4, what wassuggested that L-NIBC-modified AuCs could inhibit the amyloidosisprocess of Aβ(1-40) completely.

FIG. 4 is a qualitative experiment, but FIG. 5 is a quantitativeexperiment. The result of FIG. 5 indicates that the addition of L-NIBChad no obvious effect on Aβ(1-40) amyloidosis kinetics (panel A of FIG.5); for gold nanoparticles, when the particle diameter was greater thanor equal to 10.1 nm, the addition of L-NIBC-modified gold nanoparticlespushed forward both the growth stage and platform stage of Aβaggregation kinetics (when the concentration of gold nanoparticles was20 ppm, the growth stage of Aβ aggregation kinetics was pushed forwardto 12^(th) h, and the platform stage was pushed forward to 16^(th) h),suggesting that L-NIBC-modified gold nanoparticles could accelerate Aβaggregation (panels B and C of FIG. 5); when the diameter of goldnanoparticles was smaller than or equal to 6.0 nm (panel D and E of FIG.5), the starting time of Aβ aggregation could be delayed (when theconcentration of L-NIBC-modified gold nanoparticles was 20 ppm, thegrowth stage of Aβ aggregation kinetics was delayed to 54^(th)),suggesting that gold nanoparticles had an inhibitory effect on Aβaggregation. However, FIG. 5 indicates that even if the concentrationwas very high (20.0 ppm), the addition of L-NIBC-modified goldnanoparticles was unable to inhibit completely (i.e., no growth stageappeared, and the fluorescent curve was completely flat). On the otherhand, after addition of L-NIBC-modified gold nanoparticles, since thefluorescence emission peak of ThT locates at 515 nm, while the plasmonresonance absorption peak of L-NIBC-modified gold nanoparticles locatesnear 520 nm, the decrease of ThT fluorescent intensity observed hereshould be the partial quenching of the plasmon resonance effect of thegold nanoparticles to ThT fluorescence, but should not be attributed tothe inhibitory effect of L-NIBC-modified gold nanoparticles on Aβ(1-40)aggregation.

Panels F-H of FIG. 5 indicate that all the L-NIBC-modified AuCs couldsignificantly inhibit Aβ aggregation (the starting time of the growthstage was postponed. When the concentration of L-NIBC-modified AuCs was5 ppm, the starting time of the growth stage in aggregation kinetics of20 μM Aβ could be delayed to later than 50 h). When the concentration ofL-NIBC-modified AuCs was 10 ppm or above, Aβ aggregation could becompletely inhibited (growth stage did not appear, and the fluorescentcurve was completely flat). The minimum concentration of L-NIBC-modifiedAuCs needed for complete inhibition is relevant to the ligand type andthe diameter of AuCs. The minimum concentrations of L-NIBC-modified AuCswith sizes of 1.1 nm, 1.8 nm and 2.6 nm were 5.0 ppm, 5.0 ppm and 10.0ppm respectively. Besides, as L-NIBC-modified AuCs do not have plasmonresonance effect, they do not have quenching effect on ThT fluorescence.Therefore, the decrease in fluorescence intensity observed here wasentirely due to the inhibitory effect of L-NIBC-modified AuCs onAβ(1-40) aggregation. The quantitative results of FIG. 5 are in goodagreement with the qualitative results of FIG. 4.

This experiment indicates that: when the size of L-NIBC-modified goldnanoparticles is smaller than or equal to 6.0 nm, they have certaininhibitory effect on Aβ aggregation and fibrosis, but limitedly;L-NIBC-modified AuCs has the function of completely inhibiting Aβaggregation and fibrosis. As L-NIBC molecules per se cannot influencethe aggregation and fibrosis of Aβ (in view of panel B of FIG. 4 andpanel A of FIG. 5), this function is from AuCs, but not L-NIBC ligand.This lays a foundation for the formation of medications for Aβaggregation and fibrosis-related diseases, which can be classified asAuCs-containing substances as defined by the present invention.

This embodiment also validates the functions of AuCs modified with otherligands listed in Table 1. For example, panels A-H of FIG. 12 show theinhibitory effect of AuCs modified with CR, N-acetyl-L-cysteine (L-NAC),GSH, 1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (Cap), D-NIBC, RCor L-cysteine and D-cysteine (the dose is 10 ppm) on Aβ(1-40)aggregation and fibrosis. A similar phenomenon was observed for AuCsmodified with different ligands, and the same conclusion can be made:These ligands per se cannot influence Aβ aggregation and fibrosis, theligand-modified gold nanoparticles with a size of greater than 3 nm havea limited inhibitory effect on Aβ aggregation and fibrosis, and largergold nanoparticles even promote Aβ aggregation and fibrosis; butligand-modified AuCs have excellent inhibitory effect on Aβ aggregationand fibrosis, and when the concentration is above 5-10 ppm, effect ofcomplete inhibition can be achieved, while the minimum concentrationneeded for complete inhibition varies slightly with ligand and particlesize of AuCs. Likewise, these ligand-modified AuCs are classified intoAuCs-containing substances defined in the present invention.

Embodiment 4: Experiment of Aβ Induced AD Cell Model

The cell viability was used as an index in the experiment of thisembodiment. The test result of CCK-8 method reflected the effects ofligand-modified AuCs or gold nanoparticle samples on the toxicity ofAβ(1-40), and showed if ligand-modified AuCs or gold nanoparticles had aneuroprotective effect on the pathogenesis of amyloid proteinmisfolding. The cells used in the experiment were SH-SY5Y neuroblastomacell line. Aβ-induced AD cell model was established according to thedescription in the literature (R Liu, H. Barkhordarian, S. Emadi, C. B.Park, M. R. Sierks, Neurobiology of Disease 2005, 20, 74). Specificmethod:

1) SH-sy5y cells (cells had passed to the sixth generation) inlogarithmic growth phase were diluted with complete medium (MEM+10%FBS+1% penicillin-streptomycin) to get a cell suspension in a density of5×10⁴/mL. The suspension was inoculated 200 μL per well into a 96-wellplate, and cultivated in an incubator with 5% CO₂ at 37° C. A sample wasadded when the cells attached to the wells.

2) 100 μL ligand-modified AuCs samples or ligand-modified goldnanoparticles samples, at different particle sizes and at concentrationsof 0.04 ppm, 0.4 ppm, 4 ppm, 20 ppm, 40 ppm and 80 ppm respectively,which were dissolved by maintenance medium (MEM+2% FBS+1%penicillin-streptomycin) were added into the incubated suspensionobtained from Step 1). After incubating in the incubator for 2 h, 100 μL80 μM Aβ(1-40) was added, and then the mixture was incubated in theincubator for 24 h. In this way, the final concentrations ofligand-modified AuCs or ligand-modified gold nanoparticles were 0.01ppm, 0.1 ppm, 1 ppm, 5 ppm, 10 ppm and 20 ppm respectively, while thefinal concentration of Aβ(1-40) was 20 μM. Meanwhile, there were groupsbelow: the blank control group did not contain SH-sy5y cells, thenegative control group contained SH-sy5y cells but did not containligand-modified AuCs or ligand-modified gold nanoparticles and Aβ(1-40),the cell model control group contained SH-sy5y cells and Aβ(1-40) (finalconcentration was 20 μM) only, and the ligand control group containedSH-sy5y cells, Aβ(1-40) (final concentration was 20 μM) and L-NIBC(final concentration was 20 ppm). The culture medium was removed,maintenance medium (MEM) containing 10% CCK-8 at 100 μL/well was addedand was incubated for 4 h, the absorbance of each well at 450 nm wasmeasured to reflect the prevention and treatment effects ofligand-modified AuCs on Aβ(1-40) lesion.

L-NIBC-modified AuCs in Embodiment 2 was taken for example,L-NIBC-modified gold nanoparticles were compared with AuCs and theresults are shown in FIG. 6.

Panels A-C of FIG. 6 respectively shows the effects of L-NIBC-modifiedAuCs with a particle size of 1.1 nm, 1.8 nm or 2.6 nm at differentconcentrations on the cell viability in Aβ induced AD cell model; panelsD-F respectively shows the effects of L-NIBC-modified gold nanoparticleswith a particle size of 3.6 nm, 6.0 nm or 10.1 nm at differentconcentrations on the cell viability of the Aβ induced AD cell model.

As shown in FIG. 6, the addition of L-NIBC only did not improve cellviability. L-NIBC-modified AuCs with different sizes (the average sizeswere 1.1, 1.8 and 2.6 nm respectively) raised the cell viability fromnearly 60% to above 95% in Aβ-induced AD cell model (P values were allless than 0.05, in panels A-C of FIG. 6) even at a very low dose (0.1-1ppm for instance). L-NIBC-modified gold nanoparticles in an averagediameter of 3.6 nm raised the cell viability to some extent with theincrease of concentrations in AD cell model (panel D of FIG. 6), but notobviously (P>0.05). L-NIBC-modified gold nanoparticles in averagediameters of 6.0 nm and 10.1 nm respectively did not have effect on cellviability (panels E and F of FIG. 6). The above results indicated thatL-NIBC-modified AuCs had a significant medicinal efficacy on Aβ-inducedAD cell model, while L-NIBC-modified gold nanoparticles had no obviousefficacy.

Experiments on AuCs modified with other ligands listed in Table 1 invarious sizes were conducted in this embodiment. The results alsoindicated that ligand-modified AuCs significantly improved the cellviability in Aβ-induced AD cell model. It was indicated that AuCsmodified with different ligands had excellent therapeutic effects on ADat least at the cell model level, which could be classified into theAuCs-containing substances as defined in the present invention and beused for AD treatment.

Embodiment 5: Experiment of AD Transgenic Mouse Model Experiment 1

1) 1.0 g of AuCs modified with ligands listed in Table 1 were weighedrespectively, and dissolved in 100 mL water as stock solutions, andstored at 4° C. for future use. A small volume of the stock solutionswas taken and diluted in water before use.

2) 180 B6/J-Tg(APPswe,PSEN1de9)85Dbo/MmNju strain transgenic mice(purchased from the Model Animal Research Center of Nanjing University)were randomly divided into three groups, 60 mice per group, including acontrol group, a low dose group and a high dose group. When the micewere 100 day old, mice in the control group were fed normally everyday,mice in the low dose group were orally administered with 200 μL of 0.5g/L AuCs in water a day, and mice in the high dose group were orallyadministered with 200 μL of 2 g/L AuCs in water a day.

3) The mice in the control group, low dose group and high dose groupwere randomly divided into 7 batches respectively: When the mice were140 days, 160 days, 180 days, 200 days, 230 days, 260 days and 290 daysold respectively, maze experiment, open field experiment, and new objectrecognition experiment were adopted to research the changes of mice inlearning and memory behaviors. In the first four batches, 6 mice in eachgroup; and in the last three batches, 6-8 mice in each group(considering that there was a certain mortality rate in the feedingprocess of mice, the same below).

4) After the behavioral researches of above each batch mice, the contentof Aβ in the blood was detected: blood was collected from the orbitalvenous plexus, and the content of Aβ and Aβ aggregation were detected byserum Elisa method.

5) After the content of Aβ in the blood of above each batch mice wasdetected, the Aβ amyloid deposition distributing in the hippocampus wasdetected: the mice were anesthetized after extraction of ocular blood,they were fixed via heart perfusion. The whole brains of mice werecollected and sedimentated by gradient in sucrose. Then the brains werefreezed and sectioned. The distribution of Aβ amyloid deposits in thehippocampus was examined by immunohistochemistry.

The results showed that the ligand-modified AuCs provided in the presentinvention could significantly improve the cognitive behavior of ADtransgenic mice, and inhibit the formation of senile plaques in thebrain and the development of the disease, which may be used to treat ADas AuCs-containing substances.

Experiment 2

1. 1.0 g AuCs modified with ligands listed in Table 1 were weighedrespectively, were dissolved in 100 mL water as stock solutions storedat 4° C. for future use. A small volume of the stock solutions was takenand diluted in water before use. The stock solutions were prepared oncetwo weeks.

2. 90 B6/J-Tg(APPswe,PSEN1de9)85Dbo/MmNju strain transgenic mice(purchased from the Model Animal Research Center of Nanjing University)were randomly divided into three groups: a model control group, a lowdose group and a high dose group, 30 mice per group (considering thatthe transgenic mice of this strain had about 30% mortality in thefeeding process, in order to ensure sufficient mice in the late of thisexperiment, there were more mice in the initial than in the late of thisexperiment). When the mice were 100 days old, mice in the model controlgroup were fed normally everyday, mice in the low dose group and highdose group were orally administered with AuCs solution at a dose of 5mg/kg body weight and 20 mg/kg body weight respectively byintraperitoneal injection once every two days.

3. The cognitive behavior of mice was tested using water mazeexperiment. Morris water maze (MWM) experiment is the one to forcetested animal to swim and learn to find the platform hidden in thewater. MWM is mainly used to test the tested animal's learning andmemory abilities of the spatial positions and direction perception,which is widely adopted in the research of AD medication development andevaluation. Shorter escape latency, more times of crossing the platformafter removal, longer swimming distance in the target quadrant, andlonger staying in the target quadrant of the mice, mean that the micehave a better memory ability of spatial position and directionperception. After 150 days of administration, the behavior of each mousewas tested in Morris water maze experiment. This experimental methodrefers to the literature (C. V. Vorhees, M. T. Williams, NatureProtocols 2006, 1, 848). Details were as follows:

(1) Positioning navigation experiment: The MWM test system consisted ofa circular pool and an automatic video and analysis system. The camerasabove the pool were connected to a computer (as shown in FIG. 13). Thewater maze consisted of a circular pool in a diameter of 120 cm and aheight of 60 cm, and a platform in a diameter of 9 cm. The liquid levelwas 0.5 cm higher than the platform and the water temperature was at22±0.5° C. White pigment was used to dye water to milky white. Thepositioning experiment was used to measure the learning and memoryabilities of mice in water maze, which lasted for 4 days. As shown inFIG. 13, the water maze was divided as a cross into four quadrants inthe four directions of East (E), West (W), South (S) and North (N). Theplatform was placed in the middle of the SW quadrant, position of whichwas fixed throughout the experiment. During training, the mice weregently put into water from ½ radian in different quadrants, with headsfacing the pool wall and near the inner wall. The time that the micespent in climbing onto the hidden platform (escape latency) was recordedby a camera tracking system, or the experiment is stopped when therecord time reached 60 s. The mice were allowed to stay on the platformfor 30 s after climbing onto the platform. If the mice failed to findthe platform within 60 s (the escape latency was recorded as 60 s inthis case), the experimenters would guide the mice to climb onto theplatform and let them stay on the platform for 30 s. After theexperiment, every mouse was moved away and wiped dry gently. Each mousewas trained 4 times a day for four consecutive days, with 15-20 minintervals between training sessions.

(2) Spatial probe test: After finishing the training on the 4th day, theplatform was removed on the 5th day, the mice were gently put into thewater from the midpoint of the NE arc (the farthest point of theplatform) facing the pool wall, the 60 s movement orbits of the micewere recorded by camera, and the times of mice crossing the platform,time of stay in the target quadrant and the swimming distance in thetarget quadrant were analyzed by a software.

4. Immunohistochemistry experiment is used to detect the distribution ofamyloid deposition in the hippocampus and cerebral cortex Aβ(1-40) andAβ(1-42). The pathological deposition of Aβ outside neurons in thecerebral cortex and hippocampus is the main pathological feature of AD.Among them, Aβ(1-40) and Aβ(1-42) are important components of senileplaques in the brain, which are neurotoxic and can cause progressivecognitive dysfunction and memory loss. In this experiment, the changesof Aβ(1-40) and Aβ(1-42) plaque formation in the hippocampus andcerebral cortex were examined by immunohistochemistry.

Specific method: After the mice were administered consecutively for 100days and 150 days, 10-12 mice were taken from each group to doimmunohistochemistry of hippocampus and cerebral cortex. Here, the micewith 150 days of administration were those had completed the MWMexperiment. After the mice were anesthetized by intraperitonealinjection of 5% chloral hydrate (10 μL/g), the limbs were fixed on theexperiment table, and the chests were opened to expose the hearts fully.Pay attention to not cut the livers during thoracotomy. The leftventricles were washed with 50 mL 0.1 mol/L PBS for 5 min firstly toremove blood, and then 0.1 mol/L PBS containing 4% paraformaldehyde wasused for perfusion and fixation for 6 min After perfusion and fixation,the brains were removed and placed in 4% paraformaldehyde at 4° C. andwere fixed overnight. The tissues were dehydrated with 10%, 20% and 30%sucrose solution by gradient in turn and stored at −80° C. for futureuse. The tissues were embedded with paraffin. The midbrain hippocampusand cerebral cortex (8 pin thick) were sliced in reference to mousebrain map and used for immunohistochemical staining. The steps were asfollows: the frozen 8 μm thick slices were kept at room temperature for30 min, fixed in 4° C. acetone for 20 min, washed with PBS for threetimes (5 min each time), and then incubated in 3% H₂O₂ for 10 min toeliminate peroxidase activity. After washed with PBS for three times (5min a time), the slices were blocked with 10% normal goat serum for 40min at room temperature (the slices used for Aβ(1-42)immunohistochemistry were incubated in 10% formic acid for 10 min beforeblocking to repair antigenic activity). The serum was poured out,anti-Aβ(1-40) (ab20068, 1:20 dilution) or anti-Aβ(1-42) working fluid(ab12267, 1:200 dilution) were added into the slices, and were incubatedfor 2 h at room temperature, were washed with PBS for three times (5 mina time). Horseradish enzyme labeled streptavidin (diluted with PBS) wasadded dropwise to the secondary antibody working solution, and wasincubate at room temperature for 1 h. After washed with PBS for threetimes (5 min a time), nickel sulfate enhanced DAB blue reaction methodwas adopted for coloration for 10 min. When the positive product wasdark blue and the background was clear, it was rinsed with distilledwater for 3 times to stop coloration. After counter-stained withhematoxylin for 1 min, it was rinsed with tap water, was dried in aventilated place, and sealed with neutral gum. The number of Aβ plaquesin the entire hippocampus and cerebral cortex was observed and countedby a confocal microscope. Each sample included two slices containingboth left and right ventricles as parallel samples. The average valuewas calculated for statistical analysis. All data were processed by SPSSsoftware (SPSS 21), and undergone t test or one-way analysis ofvariance. P<0.05 meaned that the difference is statisticallysignificant.

L-NIBC-modified AuCs with an average size of 1.8 nm in Embodiment 2 wastaken for example, the result of the water maze experiment after 150days of administration was shown in FIG. 14. The result indicated thatthere was no statistical difference between the mice in the modelcontrol group and in the high dose and low dose groups in escape latencyon day 1-2 of the positioning navigation test (P>0.05, n=10-12/group)(panel A of FIG. 14). With the increase of training time, the escapelatency of the mice in the high dose group was obviously lower than thatin the model group on day 3 and day 4 (P<0.01 and P<0.05), and theescape latency in the low dose group was lower than that in the modelgroup, but no statistical difference (P>0.05, see panel A of FIG. 14).After mouse positioning navigation experiment was completed, theplatform was removed and started space search experiment. The resultsshowed that compared with the mice in the model control group, the micein the high dose group had significant increase in the times of crossingthe platform and the swimming distance in the target quadrant (P<0.05),and the time of staying in the target quadrant was also significantlyincreased, too (P=0.05). Compared with the mice in the model controlgroup, the mice in the low dose group showed increase in the times ofcrossing the platform, the swimming distance in the target quadrant andthe time of stay in the target quadrant, but no significant difference(P>0.05) (panels B-D of FIG. 14). The above results indicated that after150 days' administration of AuCs, AuCs significantly improved theability of APP/PS1 mice to learn and remember spatial position and senseof direction. And this effect was dose dependent.

The results of the immunohistochemistry experiment for detecting thedistribution of amyloid deposition of Aβ(1-40) and Aβ(1-42) in thehippocampus and cerebral cortex are shown in FIG. 15-FIG. 18.

Panels A, B and C of FIG. 15 were results of typical immunohistochemicalslices of the hippocampus and cerebral cortex of Aβ(1-40) in the highdose group, low dose group and model control group on the 100th day ofadministration. Panel D of FIG. 15 was the statistical result. Theexperimental results indicated that compared with the model controlgroup, mice in the high dose group had significantly reduced Aβ(1-40)plaques in the hippocampus (44.6±12.2%, P<0.05) on the 100^(th) day ofadministration, but not in the cerebral cortex (P>0.05). The low dosegroup did not have a significant effect on the formation of Aβ(1-40)plaques in the hippocampus and cerebral cortex (P>0.05). FIG. 16 showedthe corresponding results of Aβ(1-42). It indicated that theadministration at a high dose could significantly reduce the formationof Aβ(1-42) plaques in the cerebral cortex (reduced by 61.5111.4%,P<0.05), but not in the hippocampus (P>0.05). The administration at alow dose does not have a significant effect on the formation of Aβ(1-42)plaques in the hippocampus and cerebral cortex (P>0.05). These resultsindicated that on the 100th day of administration, AuCs had shown asignificant inhibitory effect on the formation of Aβ(1-40) and Aβ(1-42)plaques, and this effect showed an obvious dose-dependent relationship.

With the increase of administration time and age of mice, the formationof Aβ(1-40) and Aβ(1-42) plaques in the hippocampus and cerebral cortexof mice increased significantly in the model control group with 150 daysof administration compared with 100 days of administration. To bespecific, Aβ(1-40) increased 57.2±7.2% in the hippocampus (P<0.05), and49.1±19.6% in the cerebral cortex (P<0.05), and Aβ(1-42) increased74.4±7.0% in the hippocampus (P<0.05), and 65±11.1% in the cerebralcortex (P<0.05). It was suggested that the older the model mice were,the greater the influence on memory and cognitive functions might be.Panels A, B and C of FIG. 17 were results of typical immunohistochemicalslices of the hippocampus and cerebral cortex of Aβ(1-40) in the highdose group, low dose group and model control group on the 150th day ofadministration respectively. Panel D of FIG. 17 was the statisticalresult. The results indicated that in the high dose group, Aβ(1-40)decreased obviously in both the hippocampus and cerebral cortex of mice(reduced by 59.0±11.1%, P<0.05 in the hippocampus; and 36.4±4.5%, P<0.05in the cerebral cortex), while administration at a low dose did not havea significant effect on the formation of Aβ(1-40) plaques in thehippocampus (P>0.05), but significantly reduced Aβ(1-40) plaques in thecerebral cortex (reduced by 26.9±2.1%, P<0.05). It was indicated AuCshad a significant inhibitory effect on the formation of Aβ(1-40) plaqueson the 150th day. This effect also showed a dose-dependent relationship.In addition, SPSS software was used to analyze the correlation betweenthe numbers of Aβ(1-40) plaques and the times of crossing the platformin the 150-day water maze experiment. The analysis revealed that thenumbers of Aβ(1-40) plaques in the hippocampus and cerebral cortex wassignificantly negative correlative with the times of crossing theplatform (the hippocampus: R=−0.848, P<0.01; cerebral cortex: R=−0.802,P<0.05). This result further supported the correlation between thereduction of Aβ(1-40) plaques in the hippocampus and cerebral cortexinduced by AuCs administration and the improvement of memory andlearning abilities of mice induced by AuCs administration.

FIG. 18 shows the corresponding results of Aβ(1-42) upon 150-day AuCsadministration. The results indicated that the AuCs administration at ahigh dose obviously inhibited the formation of Aβ(1-42) plaques in thehippocampus and cerebral cortex (reduced by 51.1±6.7%, P<0.05 in thehippocampus; and 62.8±4.6%, P<0.05 in the cerebral cortex).Administration at a low dose did not have an obvious effect on theformation of Aβ(1-42) plaques in the hippocampus and cerebral cortex ofmice (P>0.05). It was indicated that AuCs had a significant inhibitoryeffect on the formation of Aβ(1-42) plaques on the 150th day. Thiseffect showed a dose-dependent relationship. The correlation statisticsanalysis by SPSS revealed that the numbers of Aβ(1-42) plaques in thehippocampus and cerebral cortex were significant negative correlativewith the times of crossing the platform (hippocampus: R=−0.794, P<0.05;cerebral cortex: R=−0.802, P<0.05). This result further supported thecorrelation between the reduction of Aβ(1-42) plaques in the hippocampusand cerebral cortex induced by AuCs administration and the improvementof memory and learning abilities of mice induced by AuCs administration.

In summary, AuCs could significantly improve the cognitive behavior ofAD model mice, and inhibit the formation of Aβ(1-40) and Aβ(1-42)plaques in the hippocampus and cerebral cortex, thus AuCs could inhibitthe development of the illness condition of sick mice, and be used toprevent and treat AD as AuCs-containing substances.

The AuCs modified with other ligands listed in Table 1 had similareffects, so they were not described in details here.

Embodiment 6: Experiment of In Vitro α-Syn Aggregation Kinetics

This embodiment validated the functions of ligand-modified AuCs throughin vitro α-syn aggregation kinetics experiment, and compared it with theeffect of ligand molecules on α-syn aggregation kinetics when usedalone, so as to prove that this function is from AuCs other than ligand.

Thioflavin T (abbreviation: ThT) is a dye for dyeing amyloid fibersspecifically. When it is incubated together with monomers ofpolypeptides or proteins, its fluorescence does not change much. When itencounters amyloid polypeptide or protein with a fiber structure, itwill immediately couple with the amyloid polypeptides or proteins andits fluorescence intensity will increase exponentially. For this reason,ThT is widely used as markers to monitor amyloidosis of peptides orproteins. This embodiment utilizes ThT fluorescent labeling method tomonitor the kinetics process of fibrosis aggregation of α-syn with theexistence of AuCs. The specific experiment method was as follows:

Pretreatment of α-syn monomers: Freeze-dried powder (Bachem Corp.) ofα-syn was dissolved in HFIP to obtain a 1 g/L α-syn solution. Thesolution was incubated at room temperature for 2-4 h after sealing, thenblowed to dry HFIP by high-purity nitrogen in a fume hood. Lastly thedried α-syn was dissolved in 200 μL DMSO, after sealing, the solutionwas kept in a refrigerator at −20° C. for no more than one week forfuture use. Before use, the DMSO solution of α-syn was diluted withplenty of phosphate buffer solution (PBS, 10 mM, pH=7.4) till theconcentration of α-syn reached 20 μM to obtain an α-syn PBS solution.All the α-syn PBS solutions in the experiment were prepared freshly.

Sample preparation and detection: Ligand-modified AuCs in differentconcentrations listed in Table 1 were added to 35 μM α-syn PBS solutionsrespectively, and incubated continuously in a 96-well plate at 37° C.,by ThT fluorescent labeling method, and were monitored the fluorescenceintensity by microplate reader once every 10 minutes. The kineticprocess of α-syn aggregation was characterized through the change offluorescence intensity of ThT. L-NIBC-modified AuCs with a particle sizeof 1.8 nm prepared in Embodiment 2 were taken for example as experimentgroups. L-NIBC molecules uncombined with AuCs were used as a ligandcontrol group. Four concentrations of AuNCs are adopted, which were: 0ppm (containing α-syn only, not containing AuCs or L-NIBC, as a modelcontrol group), 1.0 ppm, 5.0 ppm and 10.0 ppm respectively. L-NIBCmolecules in two concentrations were used, which were: 1.0 ppm and 10.0ppm respectively.

The results were shown in FIG. 19. The results indicated that in theincubation process of 35 μM α-syn at 37° C., ThT-labeled fluorescenceintensity increased rapidly from the 48th hour. It was demonstrated thatα-syn aggregation and fibrosis happened. This was consistent with theresult reported in the literature (V. N. Uversky, J. Li, P. Souillac, I.S. Millett, S. Doniach, R. Jakes, M. Geodert, A. L. Fink, Journal ofBiological Chemistry 2002, 277, 11970). The result of the ligand controlgroup indicated that using L-NIBC only did not have an obvious effect onkinetics of α-syn aggregation (panel A of FIG. 19). In the experimentalgroup with addition of AuCs in a low concentration (such as: 1.0 ppm and5.0 ppm), ThT-labeled fluorescence intensity decreased significantlycompared with the model control group and ligand control group withoutthe addition of AuCs, and the starting time delayed obviously (panel Bof FIG. 19). It was suggested that the addition of AuCs couldsignificantly inhibit α-syn aggregation and fibrosis. When AuCsconcentration reached 10 ppm, ThT-labeled fluorescence intensityremained near the base line without any increase throughout the 168hours of the experiment (panel B of FIG. 19). It was suggested that whenAuCs concentration was high enough, α-syn aggregation and fibrosis couldbe inhibited completely.

In this embodiment, AuCs modified with other different ligands listed inTable 1 were also studied. For example, panels C-J of FIG. 19 showed theinhibitory effects of AuCs modified with D-NIBC, CR, RC,1-[(2S)-2-methyl-3-thiol-1-oxopropyl]-L-proline (Cap), GSH,N-acetyl-L-cysteine (L-NAC), L-cysteine (L-Cys) and D-cysteine (D-Cys)(the dose were all 10 ppm) on α-syn aggregation and fibrosis. A similarphenomenon was also observed for AuCs modified with different ligands,and the same conclusion could be reached: These ligands per se could notinfluence α-syn aggregation and fibrosis, while ligand-modified AuCs hadan excellent inhibitory effect on α-syn aggregation and fibrosis. Whenthe concentration reached 10 ppm, an effect of complete inhibition couldall be achieved. The minimum concentration needed for completeinhibition varies slightly with different ligands. Likewise, theseligand-modified AuCs were classified into AuCs-containing substancesdefined in the present invention. Other AuCs listed in Table 1 hadsimilar effects. Only the concentrations of AuCs needed for completeinhibition of α-syn aggregation and fibrosis were different. It wouldnot be described in details.

Embodiment 7: Experiment of MPP+ Induced PD Cells (SH-sy5y) ModelExperiment 1

The experiment uses cell viability as an indicator; the test resultsobtained from CCK-8 method reflect the resistence efficacy ofligand-modified AuCs or gold nanoparticles against the toxic effect ofMPP⁺ (a common-used neurotoxin) in SH-sy5y nerve cell model of PD, so asto demonstrate their neuroprotection effect on PD. The MPP⁺ induced PDcell model is established according to the description in literatures(Cassarino, D S; Fall, C P; Swerdlow, R H; Smith, T S; Halvorsen, E M;Miller, S W; Parks, J P; Parker, W D Jr; Bennett, J P Jr. Elevatedreactive oxygen species and antioxidant enzyme activities in animal andcellular models of Parkinson's disease. Biochimica et biophysicaacta.1997.1362.77-86). Specific method was as follows:

1) SH-sy5y cells in the logarithmic growth phase were diluted withcomplete medium to get a cell suspension in a cell density of 5×10⁴/mL.The suspension was inoculated 200 μl per well into a 96-well plate, andcultivated in an incubator with 5% CO₂ at 37° C. A sample was added whenthe cells attached to the wells.

2) 100 μL ligand-modified AuCs samples (listed in Table 1) orligand-modified gold nanoparticles samples with different particle sizesand different concentrations, were dissolved in maintenance medium, wereadded as the first groups to make the final concentrations be 0.01 ppm,0.1 ppm, 1 ppm, 5 ppm, 10 ppm and 20 ppm respectively. The first groupswere the administration groups. After 2 hours of pretreatment onligand-modified AuCs or gold nanoparticles, MPP⁺ (final concentrationwas 1 mM) was added to the administration groups and the cell controlgroup respectively, simultaneously, a blank control group was the onenot containing SH-sy5y cells, a negative control group was the onecontaining SH-sy5y cells but without AuCs or gold nanoparticles andMPP⁺, a cell control group was the one containing SH-sy5y cells and 1 mMMPP⁺ only, and a ligand control group was the one containing SH-sy5ycells and 1 mM MPP⁺ and corresponding ligand molecules (finalconcentration was 20 ppm), then the samples in every groups wereincubated at 37° C. for 24 h, were centrifuged to remove the culturemedium, 100 μl, maintenance medium containing 10% CCK-8 was added intoeach well, and continued to be incubated for 4 h, and then theabsorbances of each well were measured at 450 nm to reflect thepre-protective and curative effects of ligand-modified AuCs against MPP⁺lesion.

The same steps were adopted to carry out the experiments for AuCs andgold nanoparticles modified with different ligands. The resultsindicated that the ligand-modified AuCs provided in the presentinvention had a neuroprotective effect on PD. This effect was alsooriginated from AuCs, rather than the ligand. They can be used assubstances containing AuCs to resist PD.

Experiment 2

The experiment uses cell viability as an indicator. The test resultsobtained from CCK-8 method reflect the resistence efficacy ofligand-modified AuCs or gold nanoparticles against the toxic effect ofMPP⁺ (a common-used neurotoxin) in SH-sy5y nerve cell model of PD,demonstrate their neuroprotection effect on PD. The MPP⁺ induced PD cellmodel is established according to the description in the reference (D.S. Cassarino, C. P. Fall, R. H. Swerdlow, T. S. Smith, E. M. Halvorsen,S. W. Miller, J. P. Parks, W D. Jr. Parker, J. P. Jr. Bennett,Biochimica et Biophysica Acta 1997, 1362, 77). Specific method:

1) SH-sy5y cells in the logarithmic growth phase were diluted withcomplete medium to get a cell suspension in a cell density of 5×10⁴/mL.The suspension was inoculated 200 μl per well into a 96-well plate, andcultivated in an incubator with 5% CO₂ at 37° C. A sample was added whenthe cells attached to the wells.

2) 100 μL ligand-modified AuCs samples (listed in Table 1) orligand-modified gold nanoparticles samples with different particle sizesand different concentrations, were dissolved in maintenance medium, wereadded as the first groups to make the final concentrations be 1 ppm, 5ppm, 10 ppm, 40 ppm respectively. The first groups were theadministration groups. After 2 hours of pretreatment on ligand-modifiedAuCs or gold nanoparticles, MPP⁺ (final concentration was 1 mM) wasadded to the administration groups and the cell control grouprespectively, simultaneously, a blank control group was the one notcontaining SH-sy5y cells, a negative control group was the onecontaining SH-sy5y cells but without AuCs or gold nanoparticles andMPP⁺, a cell control group was the one containing SH-sy5y cells and 1 mMMPP⁺ only, a AuCs control group was the one containing 100 ppm AuCs butwithout MPP⁺, and a ligand control group was the one containing SH-sy5ycells and 1 mM MPP⁺ and corresponding ligand molecules (finalconcentration was 20 ppm), then the samples in every groups wereincubated at 37° C. for 24 h, were centrifuged to remove the culturemedium, 100 μL maintenance medium containing 10% CCK-8 was added intoeach well, and continued to be incubated for 4 h, and then theabsorbances of each well were measured at 450 nm to reflect thepre-protective and curative effects of ligand-modified AuCs against MPP⁺lesion.

The experimental results of L-NIBC-modified AuCs or gold nanoparticleswere taken for example, as shown in FIG. 20. The results indicated thatafter 24 hours of cultivation, the cell viability of the AuCs controlgroup, adding 100 ppm AuCs without MPP⁺, increased to 108.5±7.1%relative to the blank control group (set as 100%) (P<0.01), suggestingthat AuCs were nontoxic. The cell viability of the model control group,adding 1 mM MPP⁺ but without AuCs, decreased to 65.1±4.0% (v.s. theblank control group, P<0.01), the cell viability of the ligand controlgroup was 61.5±3.8% (v.s. the blank control group, P<0.01), suggestingthat ligand only did not raise the viability of MPP⁺ introduced cellmodel. While the cell viability of the administration group, adding 1ppm, 5 ppm, 10 ppm and 40 ppm of AuCs respectively, increased to97.9±2.8% (v.s. the model control group, P<0.01), 99.7±4.0% (v.s. themodel control group, P<0.001), 95.3±1.7% (v.s. the model control group,P<0.01) and 93.2±0.4% (v.s. the model control group, P<0.01)respectively, suggesting that the ligand-modified AuCs provided in thepresent invention had a protective effect on nerve cells in PD, and thiseffect was also originated from AuCs rather than the ligand. On theother hand, the corresponding gold nanoparticles with the same liganddid not help to improve the viability of the model cells at allexperiment concentrations, indicating that the gold nanoparticles couldnot be used as a medicine for the prevention and treatment of PD.

The same steps were adopted to carry out the experiments for the AuCsmodified with different ligands listed in Table 1. The effects weresimilar, so it will not be described in details here.

Embodiment 8: Experiment of MPP⁺ Induced PD Cell (PC12) Model

The model of MPP⁺ (100 mM) induced apoptosis of PC12 cells was used,combining with the flow cytometry technique to observe the protectiveeffect of AuCs on MPP⁺-induced cell injury and apoptosis in thisexperiment. Specific method: A blank control group was the one withoutaddition of MPP⁺ and AuCs, an MPP⁺ model group was the one with additionof MPP⁺ only, an AuCs control group was the one with addition of AuCsonly, and an experiment group was the one with addition of both MPP⁺ andAuCs. In a experiment group, the solution of L-NIBC-modified AuCs withan average particle size of 1.8 nm was added into PC12 cell suspension(final concentration of AuCs was 20 ppm) half an hour later, MPP⁺ wasadded, and the mixture was incubated for 24 hours, Annexin V-FITC/PIcell apoptosis detection kit (purchased from Roch) and FACSCalibur flowcytometer were used to detect the growth activity and apoptosis of thecells, and the data was acquired and analyzed by CellQuest Pro.

The experiment results were shown in FIG. 21. The cell cytometrydetection results indicated that after incubation with MPP⁺ for 24 h, asindicated in the cell cytometry detection, the cell apoptosis percentageof the blank control group, without addition of MPP⁺, was 23.5±2.8%.When 20 ppm AuCs alone was coincubated with PC12 cells, the cellapoptosis percentage was 28.47±3.2%, which did not show a significantdifference from the blank control group, suggesting that AuCs did nothave apparent cytotoxic effect. The cell apoptosis percentage of theMPP⁺ model group was 49.5±10.1%, which increased significantly comparedwith the blank control group (P<0.001). When PC12 cells were incubatedwith AuCs for a half hour before the addition of MPP⁺, and werecoincubated for 24 h, the cell apoptosis percentage decreased to35.9±2.2%, compared with the MPP⁺ model group, the cell apoptosisreduced significantly (P<0.05).

The same steps were adopted to carry out the experiments for the AuCsmodified with different ligands listed in Table 1. The effects weresimilar, so it would not be described in details here.

The results of Embodiment 7 and Embodiment 8 jointly indicated that AuCscould efficiently improve the cell viability and significantly inhibitthe cell apoptosis of MPP⁺ induced PD cell model.

Embodiment 9: Experiment of MPTP Induced PD Mouse Model Experiment 1

Experimental animals: 80 C57b1/6 male mice, 8 weeks old, body weight25-30 g; 3 mice in each cage, were all raised in an environment of roomtemperature 22-27° C., 12 h circadian rhythms, with eating and drinkingfreely, and acclimated for 7 days.

MPTP induced PD mice model: Mice were randomly divided into four groups,20 mice per group, including a blank control group, an AuCs normalcontrol group, an MPTP model group and an AuCs treatment group. In theMPTP model group and the AuCs treatment group, 20 mg/kg (free base) MPTPwas injected subcutaneously once every 2 h for four times. In the blankcontrol group, 20 mg/kg normal saline was injected subcutaneously onceevery 2 h for four times. 8 h later than the last injection, in theblank control group and the MPTP model group, 10 μL normal saline wasinjected intravenously every day, while in the AuCs normal control groupand the AuCs treatment group, 10 μL the normal saline solutions ofligand-modified AuCs listed in Table 1 (AuCs concentration 10 g/L) wereintraperitoneally injected respectively every day. The injection lastedfor 7 days. The animals were put in feeding boxes with clean padding,and water and food freely.

Behavioral test: Rotarod test, rotarod test requires animals to maintainbalance and move continuously on a roller. It is a test widely used totest the motor coordination. Roller diameter is 6 cm and the rotationspeed is 20 rpm. After the animals adapted to the roller for five times,the test was started at an interval of 1 min. The latency to fall offthe rotarod was recorded consecutively for 5 times, and their averagevalue was calculated.

Neurotransmitter determination: After behavioral test, the animals weresacrificed, and the mice striatum was taken and stored at −80° C. Duringmeasurement, the striatum was treated with 10 μL/mg (striatum)homogenate (0.1M perchloric acid, 0.1 mM EDTA-2Na), was cracked byultrasonication in an ice bath for 30 min, was centrifuged in arefrigerated centrifuge at 10000 r/min for 10 min. The supernatant wastaken and filtered with a 0.25 μm filter, was injected into the liquidchromatographic column of HPLC. The level of dopamine (DA) transmitterand its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) andhomovanillic acid (HVA) in striatum were detected by the highperformance liquid phase system in laboratory. The chromatographiccolumn must be maintained with a freshly prepared mobile phase for 2 hbefore each test. HPLC conditions: flow rate: 1 mL/min; columntemperature 30° C.; fluorescence detector excitation and absorptionwavelengths were 280 nm and 330 nm respectively.

Determination of tyrosine hydroxylase: The brain tissues were taken outand fixed in 4 wt % PFA+2 wt % sucrose for 4-6 h, then were soaked in a30 wt % sucrose solution, were embed with OCT after the tissues sinkedto the bottom, were continuous coronal sectioned by frozen slicer, werestained in ABC (Avidibiotin-peroxidase complex) method, took out thefrozen tissues of substantia nigra and sectioned them, were stained inTH and colored in diphenylamine, were observed under a microscope andtaken photos.

The results indicated that the ligand-modified AuCs provided in thepresent invention could significantly improve the motor behaviors ofMPTP induced PD model mice, increase the numbers of dopaminergicneurons, improve the intracerebral level of dopamine neurotransmitters.They could be used as substances containing AuCs to treat PD.

Experiment 2

Experimental animals: 80 C57bl/6 male mice, 8 weeks old, body weight25-30 g; 3 mice in each cage, were all raised in an environment of roomtemperature 22-27° C., 12 h circadian rhythms, with eating and drinkingfreely, and acclimated for 7 days.

MPTP induced PD mice model: Mice were randomly divided into four groups,20 mice per group, including a blank control group, AuCs control groups(based on the dose of AuCs, they were classified into low dose group andhigh dose group), an MPTP model group, AuCs treatment groups (based onthe dose of AuCs, they were classified into low dose group and high dosegroup). In the MPTP model group and AuCs experiment groups, 30 mg/kg(free base) MPTP was injected intraperitoneally respectively once a dayfor 7 days consecutively. In the blank control group, 30 mg/kg normalsaline was injected subcutaneously once a day for 7 days consecutively.In the low dose AuCs control group and the low dose AuCs treatmentgroup, 100 μL normal saline solutions of 1 g/L L-NIBC-modified AuCs withan average particle size of 1.8 nm were injected intraperitoneallyrespectively once a day, while in the high dose AuCs control group andthe high dose AuCs treatment group, 100 μL, normal saline solutions of 4g/L L-NIBC-modified AuCs with an average particle size of 1.8 nm wereinjected intraperitoneally respectively once a day, for 7 daysconsecutively. The animals were put in a feeding box with clean padding,and water and food freely.

1. Behavioral Testing:

(1) Spontaneous locomotor activity test: The animals were transferredfrom the cages to the autonomous activity detector. After the animalsadapted to the new environment for 5 min, their spontaneous activitiesand the changes were recorded within 5 min. The activity distance andmovement speed of the animals within 5 min were used to measure theactivity of animals.

(2) Swim test: Refer to Donnan's test method (G. A. Donnan, G. L.Willis, S. J. Kaczmarezyk, P. Rowe, Journal of the Neurological Science1987, 77, 185), the test mice were put into a Morris water tank. Thedepth of water was 60 cm and the temperature was 22° C. The swimmingdistance and swimming time of the animals within 10 min was recorded tomeasure the activity of the animals.

(3) Rotarod test: Rotarod test requires animals to maintain balance andmove continuously on a roller. It is widely used to test the motorcoordination. Roller diameter is 6 cm and the rotation speed is 20 rpm.After the animals adapted to the roller for five times, every test wasperformed with an interval of 1 min. The latency to fall off the rotarodwas recorded. The test was conducted for 5 times consecutively and theiraverage values were calculated.

2. Immunohistochemical Detection in the Striatum and the SubstantiaNigra:

After behavioral tests, 5 mice in each group were taken to conductimmunohistochemical detection in substantia nigra and striatum. Afterabdominal anesthesia by 1 mL 0.5% pentobarbital sodium, the chests wereopened, 15 mL of 0.9% normal saline was used to rinse the blood fromaorta at first, and then 100 mL 0.1 mol/L of phosphate buffer solution(PBS, pH 7.2) containing 4% paraformaldehyde was used for perfusion(first fast then slowly) and fixation for 1 h. The brains were taken outafter perfusion and fixation, were put into 4% paraformaldehyde, wereembed with paraffin in reference to mice brain map, substantia nigra andstriatum were coronal sectioned. The thickness of brain slices was 3μm/section. The obtained brain slices were used in immunofluorescence,hypersensitive two-step immunohistochemistry and other experiments.immunohistochemical staining steps were as follows: The obtained brainslices were in 0.3% H₂O₂ methanol solution (30% H₂O₂ 1 mL+methanol 80mL+PBS 19 mL) for 30 min, in 0.3% Triton X-100 PBS for 30 min, weresoaked into mice anti-tyrosine hydroxylase (TH) monoclonal antibody(1:200) or IBa1 (dilution ratio 1:250) and incubated for 48 h (4° C.),were soaked into biotinylated rabbit anti-mouse secondary antibody(1:500) and incubated for 2 h (room temperature). They were rapidlyrinsed with distilled water and colored by nickel ammonium sulfateenhanced DAB blue reaction method for 20˜30 min. When the positiveproduct was dark blue and the background was clear, the brain sliceswere rinsed with distilled water for 3 times to stop coloration. Aftereach of the above steps, the brain slices should be rinsed by 0.01 mol/LPBS for three times and for 10 min each time. And the primary antibodyused here was diluted with PBS containing 1% bovine serum and 0.3%Triton X-100, and the complex of secondary antibody and ABC were dilutedwith PBS. Adherence of brain slices, dehydration and sealing withtransparent neutral gum were conducted.

3. Protein Immunoblotting (WB) Assay:

Tyrosine hydroxylase (TH) is a key enzyme in the biosynthetic pathway ofdopamine (DA), and immunohistochemistry of TH can show changes inDA-ergic neurons in substantia nigra and striatum (D. Luo, J. Zhao, YCheng, S. M. Lee, J. Rong, Molecular Neurobiology 2017, DOI:10.1007/s12035-017-0486-6). After behavioral tests, five mice were takenfrom each group to conduct striatum WB detection, the needed braintissues were take out on ice, were cracked in RIPA lysate, werecentrifuged at 4° C. and 12000 g for 30 min to homogenate, proteins wereextracted to prepare samples, SDS-polyacrylamide gel electrophoresis at55V-60V for 4.5 h, and the proteins were transferred to the membrane bysemi-thy method at constant current of 60 mA for about 1.5 h. Themembrane was blocked with 5% skimmed milk at room temperature for 1 h;TBST-diluted rabbit TH antibody (dilution ratio 1:300) was added, stayedovernight at 4° C.; the antibody was recovered, the membrane was washedwith TBST for three times, 10 min each time; TBST-diluted IRDye R 680RDGoat anti-Rabbit (dilution ratio 1:3000) was added; the membrane waswashed with TBST for three times, 10 min each time; protein signals werescanned by dual-color infrared laser imaging system.

The test results of the spontaneous locomotor activity of mice wereshown in FIG. 22. Three to five minutes after administration of MPTP,mice showed tremor, decreased movement, arched back, hind limb opening,gait instability, vertical tail, and vertical hair. Individual epilepticseizures occurred after about 30 to 60 minutes. The above symptomsgradually reduced, and the mice returned to normal after 24 hours.However, with the increase of the number of administrations, the acuteresponse was relieved, but after 24 hours, the performance of exercisedecreased, gait instability and slow response became more obvious. Aftercontinuous administration of MPTP for seven days, the spontaneousactivity distance and movement speed of the mice were significantlylower than those of the blank control group (P<0.01), showing thesymptoms of bradykinesia. The administration of AuCs alone had nosignificant effect on the spontaneous activity distance and movementspeed of normal mice (panels A and C of FIG. 22). The combinedadministration of AuCs (high dose) in mice of the MPTP model couldsignificantly increase the spontaneous activity distance and movementspeed of the mice (panels B and D of FIG. 22), suggesting that AuCsplayed a significant role in improving the spontaneous locomotoractivity of MPTP model mice. Compared with MPTP model group, thedifference was significant (spontaneous activity distance: P<0.05;movement speed: P<0.01).

The results of the swim test of mice were shown in FIG. 23. After sevendays of continuous MPTP injection, the mice were placed in a water tankto do the swimming test. Longer swimming time and farther swimmingdistance of mice indicate the mice had better motor coordination oflimbs. The blank control group and the AuCs control group didn't havesignificant effect on the swimming time and swimming distance of mice(panels A and C of FIG. 23). Compared with the blank control group, MPTPmodel group showed significant decrease of swimming distance in 10 min(P<0.05), and significant decrease of the swimming time in the watertank (P<0.05), suggesting that MPTP significantly reduced the motorability of swimming for the mice. Compared with the MPTP model group,the AuCs treatment group with administration of AuCs (high dose) andMPTP showed increase of swimming distance (P<0.05) and significantincrease of swimming time (P<0.05) (panels B and D of FIG. 23),suggesting that AuCs significantly improved MPTP-induced motor behaviordisorder of swimming for the mice.

The results of the mouse rotarod test were shown in FIG. 24. After themice were injected with MPTP consecutively for 7 days, they weresubjected to the rotarod test. In the blank control group administratedwith normal saline, the latency to fall off the rotarod and thepercentage of mice falling off the rotarod were 12.1±4.6 min and33.3±1.5% (panels A and C of FIG. 24). Compared with the blank controlgroup, the rod drop latency of the mice in the MPTP model group wasshortened significantly to 5.5±3.7 min, and the rod drop percentageincreased significantly to 83.3±3.4%. It was indicated that after theadministration of MPTP, the motor coordination of mice decreased and themice, could not grasp the rod stably and were liable to falling off therotarod (panels B and D of FIG. 24). The administration of AuCs alonehad no significant effect on the fall latency of mice (image A of FIG.24), but during long-time roller motion, the percentage of mice fallingoff the rotarod increased significantly (P<0.001 v.s. the blank controlgroup). It was indicated that the self-administration of AuCs had acertain effect on the roller behavior of mice (panel C of FIG. 24). Butcompared with the MPTP model group, the AuCs treatment group withadministration of both MPTP and AuCs showed obvious increase of latencyto fall off the rotarod (low dose group: P<0.01; high dose group:P<0.05), and significant decrease of the percentage falling off therotarod (P<0.001 for both the high dose group and the low dose group).The results were shown in panels B and D of FIG. 24. This indicated thatAuCs have the effect to improve MPTP-induced motor coordinationdysfunction.

The results of immunohistochemical detection in substantia nigra andstriatum and WB detection of striatum were shown in FIG. 25. Comparedwith the blank control group, the MPTP model group showed obviousdecrease in the number of TH immunopositive neurons (i.e., DA-ergicneurons) in substantia nigra with shrinkage of residual neurons andreduction or disappearance of neurites, and reduction of THimmunopositive neurons in striatum and decrease of nerve fiber density.The results of WB analysis indicated that DA-ergic neurons in striatumreduced to 55.8˜5.6% (blank control group: 100%) (v.s. the blank controlgroup, P<0.01, see panel C of FIG. 25). The administration of AuCs alonedidn't have significant effect on TH immunopositive neurons and thedensity of nerve fibers in substantia nigra and striatum (panels A and Bof FIG. 25). The combined administration of AuCs and MPTP couldsignificantly inhibit MPTP induced down-regulation of TH immunopositiveexpression in cells and neurofilaments of substantia nigra and striatum.The results of WB analysis indicated that when low dose AuCs wereadopted, the ratio of DA-ergic neurons in striatum was 65.6±6.3% of theblank control group (P<0.01, v.s. the MPTP model group, see panel C ofFIG. 25), and when high dose AuCs were adopted, the ratio of DA-ergicneurons in striatum reached 84.7±4.5% of the blank control group(P<0.001, v.s. the MPTP model group). The results indicate that AuCs hada significant effect on resisting MPTP cytotoxicity, and showed aremarkable protective effect against DA-ergic neuron loss in substantianigra and striatum.

The same method was also adopted to conduct experiments using the AuCsmodified with different ligands listed in Table 1. The effects weresimilar, so it would not be described in details here.

The above results indicated that the ligand-modified AuCs provided inthe present invention could significantly improve the spontaneouslocomotor activity, motor ability and body coordination ability of MPTPinduced PD model mice, and had a significant protective effect againstDA-ergic neuron loss in substantia nigra and striatum, indicating thatsubstances containing AuCs can be used to treat PD.

Embodiment 10: Biosafety Evaluation

1. SH-sy5y cell line was adopted to evaluate the biosafety of thesubstances containing AuCs at the cell level.

Specific method: SH-sy5y cells in the logarithmic growth phase of cells(cells at passage 6) were collected. The concentration of cellsuspension was adjusted, and added 100 μL into each well. The cells wereplated, and the cell density was adjusted to 1000-10000 per well. Thecell cuture plates (the marginal wells of 96-well plates were filledwith cell culture medium) were put in a cell incubator and incubated ina 5% CO₂, 37° C. environment for 24 h so that the cells attached to thewall. The 96-well plates were taken out, and then put in a biosafetycabinet after disinfection by alcohol. The original cell culture mediumwas sucked out, and then solutions of ligand-modified AuCs listed inTable 1 were added, which were diluted with cell culture medium toobtain a final concentration of 1 ppm, 10 ppm, 50 ppm, 100 ppm, 200 ppmand 500 ppm, respectively. An equal volume of fresh cell culture mediumwas added to the control group (no AuCs). And put it in a cellincubator, and incubated for 48 h. 6 duplicate wells were set for eachof the experiment group and the control group. After 48 h of incubation,the culture medium was removed by centrifuging, then washed with PBS for2-3 times. 100 μL fresh culture medium and 20 μL methyl thiazolyltetrazolium (MTT) solution (5 mg/ml, i.e., 0.5% MTT) were added to eachwell, and continued to be cultivated for 4 h. The cultivation wasterminated, the 96-well plate was taken out, and centrifuged (1000r/min) for 10 min. The supernatant was sucked out, and 200 μL DMSO wasadded to each well, and put on a shaking table, and oscillated at a lowspeed for 10 min till the color in the wells was uniform and crystal wasfully dissolved. The absorbance of each well was measured at 490 nm bymicroplate reader. The above operations must be conducted in a sterileenvironment. Except detection, all the steps were completed in abiosafety cabinet. The experimental supplies must be disinfected in anautoclave before use.

L-NIBC-modified AuCs in Embodiment 2 were taken for example, the resultswere shown in FIG. 26 where panels A-C showed the effects of AuCs withparticle sizes of 2.6 nm, 1.8 nm or 1.1 nm and at final concentrationsof 1 ppm, 10 ppm, 50 ppm, 100 ppm, 200 ppm or 500 ppm on SH-sy5y cellviability. It was indicated that at a fairly high concentration (suchas: 100 ppm), the addition of L-NIBC-modified AuCs almost didn't haveany influence on cell viability. At a higher concentration (such as: 200and 500 ppm), the addition of L-NIBC-modified AuCs would cause slightcell injury (cell death rate is less than 20%). Since 100 ppm was muchhigher than the lowest effect concentration of the AuCs (0.1˜1 ppm orlower), it could be concluded that L-NIBC-modified AuCs had high safetyat the cell level.

Other ligand-modified AuCs with different sizes listed in Table 1 alsohad similar effects. They would not be described in detail here.

2. Adopt Mouse Acute Toxicity Study to Evaluate the Acute Toxicity ofSubstances Containing AuCs.

Specific method: For different ligand-modified AuCs listed in Table1(L-NIBC-modified AuCs with an average diameter of 1.8 nm in Embodiment2 was taken for example), 60 adult mice were taken, and divided intofour groups with 15 mice in each group, which were: a control group andthree experiment groups. In the control group, mice were fed normally,while in the three experiment groups, mice were fed with AuCs by oraladministration (by gavage) at a dose of 0.1 g/Kg body weight, 0.3 g/Kgbody weight and 1 g/Kg body weight a day respectively for one week underthe condition of normal diet. The mice were fed normally for 30 daysafter the feeding of AuCs was finished. Abnormal responses of the micewere observed.

In the mice experiment, the ingestion of AuCs with different sizes atthree concentrations had no influence on the survival and activity ofmice. Even though for high dose intake of 1 g/Kg body weight, the micestill remained healthy.

Other ligand-modified AuCs listed in Table 1 also had similar results.They would not be described in details here. Based on the above results,it could be concluded that AuCs were very safe.

Embodiment 11: Tissue Distribution and Metabolic Distribution of theCuCs-Containing Substances in Mice Experiment 1

Operating steps: 80 mice were randomly divided into four groups, 20 micein each group, and fed with ligand-modified AuCs listed in Table 1 byoral administration (by gavage) at doses of 100 mg/kg, 20 mg/kg, 5 mg/Kgand 1 mg/kg respectively in the groups. After feeding of AuCs, the 20mice in each group were randomly divided into 4 subgroups with 5 mice ineach subgroup. They were scarified at the time points of 2 h, 6 h, 24 hand 48 h respectively after feeding. Heart, liver, spleen, lung, kidneyand brain tissues were taken separately. Each tissue was weighed, and 2mL water was added to homogenize, and then 2 mL aqua regia was added andmixed under vortex, and oscillated for 72 h on an oscillator. 2 wt %nitric acid solution was added to a final volume of 10 mL, andcentrifuged at 15000 rpm for 15 min. 4 mL of supernatant was sucked, andthe content of gold element in the tissue fluid was measured by atomicabsorption spectrometry.

The results indicated that AuCs could pass through the blood-brainbarrier and reached the brain. They could be excreted out of the bodyover time, so they didn't have obvious accumulation in the body.Therefore, the substances containing AuCs provided in the presentinvention had a good prospect in the application of preparation ofmedication treating AD or PD.

Experiment 2

Operating steps: 80 mice were randomly divided into four groups with 20mice in each group, and injected intraperitoneally with ligand-modifiedAuCs listed in Table 1. The doses of AuCs (L-NIBC-modified AuCs with anaverage diameter of 1.8 nm were taken for example) in each group were100 ppm, 20 mg ppm, 5 ppm and 1 ppm of mouse body weight respectively.After injection of AuCs, the 20 mice in each group were randomly dividedinto 4 groups, 5 mice in each group. They were scarified at time pointsof 2 h, 6 h, 24 h and 48 h respectively after feeding. Heart, liver,spleen, lung, kidney and brain tissues were taken separately. Eachtissue was weigh, and 2 mL water was added to homogenize, then 2 mL aquaregia was added and mixed under vortex, and oscillated for 72 h on anoscillator. 2 wt % nitric acid solution was added to a final volume of 5mL, and centrifuged at 15000 rpm for 15 min. 1 mL supernatant wassucked, and the content of gold element in the tissue fluid was measuredby atomic absorption spectrometry.

Above steps were adopted to carry out experiments for AuCs modified withother ligands listed in Table 1.

The results indicated that after 2 h, the content of gold element in thebrain reached 1%-10% of initial concentration. After 6 h, the content inthe brain could be maintained at a similar level. After 24 h, thecontent in the brain decreased significantly. At hour 48 h, the contentdecreased to near or below the detection limit except for the specimensat a dose of 100 ppm. The above results indicated that substancescontaining AuCs also had good biosafety at the animal level, which canpass through the blood-brain barrier, and had no obvious accumulation inthe body.

In summary, the above experiment results illustrated the followingpoints (the “gold nanoparticles” and “AuCs” mentioned below all refer tothe cases with ligand modification):

(1) In the experiment (Embodiment 3) for Aβ aggregation in vitro, it wasfound that the effect of gold nanoparticles on Aβ aggregation kineticswas related to size. When the particle diameter was greater than orequal to 10.1 nm, the addition of gold nanoparticles could acceleratethe aggregation of Aβ, and when the particle size was smaller than orequal to 6.0 nm, the aggregation of Aβ was inhibited, but completeinhibition of Aβ aggregation could not be achieved. However, when AuCswere used (average diameter is less than 3 nm), all the AuCs couldsignificantly inhibit Aβ aggregation in vitro, and this effect wasrelated to the concentration of AuCs. When the concentration of AuCsreached 5-10 ppm, the aggregation of Aβ could be inhibited completely,and the minimum concentration required for complete inhibition wasrelated to the type of ligand and the diameter of AuCs. In the in vitroexperiment (Embodiment 6) for inhibition of α-syn aggregation, it wasalso found that AuCs had the same effect of complete inhibition of α-synaggregation and fibrosis.

(2) In Aβ induced cell AD model and MPP⁺ induced cell PD modelexperiment (Embodiment 4, Embodiment 7 and Embodiment 8), it was foundthat gold nanoparticles with small particle sizes (for example, goldnanoparticles with an average diameter of 3.6 nm or 6.0 nm) didn't havea significant effect on the improvement of cell viability of Aβ inducedcell AD model and MPP⁺ induced cell PD model, suggesting that goldnanoparticles didn't show obvious medicinal efficacy on AD and PD at thecell level, so they could not be directly used as active ingredient toprepare medication treating AD or PD. However, for differentligand-modified AuCs with different sizes used in the present invention(the average diameter was less than 3 nm), it was found that a very lowdose of AuCs (such as: 0.1-1 ppm) could still raise the cell viabilityof the two models from 50%-65% to above 95%. It indicated that at thecell level, the medicinal efficacy of AuCs was significant. As theligands had no effect on Aβ aggregation and both the two cell models(Embodiments 4, 7 and 8), it could be concluded that the medicinalefficacy of AuCs was from AuCs themselves. This offered a new approachfor the application of AuCs.

(3) Further, the transgenic AD mice model and the MPTP induced PD micemodel (Embodiment 5 and Embodiment 9) were used in the present inventionto further verify the medicinal efficacy of AuCs, indicating that theAuCs played a significant role in improving mouse's cognitive behavioralability and motor behavioral ability, inhibiting the formation of senileplaques in the brain and inhibiting the specific apoptosis of DA-ergicneurons induced by MPTP in substantia nigra and striatal, and could beused as preventive or therapeutic medication for related diseases.

(4) In the experiment for further evaluation of biosafety (Embodiment10), when AuCs at a concentration of 100 ppm (by weight) wereco-cultured with nerve cells, they didn't have an obvious influence onthe viability of cells; when the concentration exceeded 100 ppm (muchhigher than the lowest effect concentration of the AuCs), the cellviability decreased slightly. As the lowest effect concentration of AuCs(0.1-1 ppm) was much lower than 100 ppm, it could be concluded that AuCshad excellent biosafety at the cell level. In the mouse acute toxicitytest, it had been found that a dose of 1 g/Kg body weight (equivalent to1000 ppm) AuCs administered once a day for seven days consecutively didnot cause adverse effect. In the study of in vivo distribution andpharmacokinetics in mice (Embodiment 11), the content of gold element inthe brain reached 1%-10% of the initial concentration after 2 h. After 6h, the content in the brain maintained at a similar level. After 24 h,the content in the brain decreased significantly. At 48 h, the contentdecreased to below the detection limit except for the specimens at adose of 100 ppm. The above results indicate that a substance containingAuCs also has good biosafety at the animal level, could pass through theblood-brain barrier, and has no obvious accumulation in the body, so ithad a good prospect in the application in preparation of medicationtreating AD or PD.

(5) Compared with current technology, the ligands used in the presentinvention were not specifically designed for the aggregation behaviorsof AP and α-syn, and the contrast experiment indicated that the ligandsused had no obvious effect on the aggregation of Aβ and α-syn(Embodiment 3). But since the size of AuCs was smaller than the size ofthe protein itself, the aggregation of Aβ and α-syn could be greatlyinhibited by the combination of the size effect and the weak molecularinteractions. The excellent efficacies in Aβ induced AD cell model andtransgenic animal model further confirmed the feasibility of substancescontaining AuCs in the preparation of medication for the treatment ofAD. In addition, the excellent efficacies of substances containing AuCsin MPP⁺ induced PD cell model and MPTP induced PD animal model indicatedthat substances containing AuCs also had broad application prospects inthe preparation of medication treating other neurodegenerative diseases.Moreover, since MPP⁺ induced PD cell model and the MPTP induced PDanimal model didn't involve protein fibrosis, but acted on deepermechanisms including function of signal transduction related to energymetabolism and neurotransmitter metabolism of nerve cells, it could bespeculated that substances containing AuCs could not only affect proteinfibrosis but also influence the process of neurodegenerative diseases ata deeper level. It will be of great significance to the research anddevelopment of new medication for neurodegenerative diseases.

INDUSTRIAL APPLICABILITY

The substances containing AuCs provided in the present invention canimprove mouse's cognitive behavior and motor behavior abilities andinhibit the formation of senile plaques in the brain in AD transgenicmouse model and MPTP induced PD mouse model, and have good biosafety atthe animal level. They are suitable for industrial applications.

The invention claimed is:
 1. A method for treating a sr bjec withAlzheimer's disease, said method comprising: administrating apharmaceutical composition into the subject with Alzheimer's disease inan effective amount; wherein said pharmaceutical composition comprisinga substance containing gold clusters (AuCs); wherein said substancecomprises: AuCs; a ligand Y coating the AuCs externally, wherein theligand Y is a thiol-containing compound; and a pharmaceuticallyacceptable recipient; wherein the AuCs has a gold core diameter smallerthan 3 nm; wherein the thiol-containing compound is one selected fromthe group consisting of: N-isobutyryl-L-cysteine (L-NIBC), andN-isobutyryl-D-cysteine (D-NIBC).
 2. The method according to claim 1,wherein the AuCs has a gold core diameter of 0.5-2.6 nm.